Dialkylphenyl compounds having beta2 adrenergic receptor agonist and muscarinic receptor antagonist activity

ABSTRACT

This invention relates to compounds of formula I: 
     
       
         
         
             
             
         
       
         
         
           
             wherein R 1  and R 2  are as defined in the specification, or a pharmaceutically acceptable salt or solvate or stereoisomer thereof. The invention also relates to pharmaceutical compositions and combinations comprising such compounds, processes and intermediates for preparing such compounds, and methods of using such compound to, for example, treat pulmonary disorders, such as chronic obstructive pulmonary disease and asthma.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/794,702, filed on Apr. 25, 2006; the entire disclosure of which isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to novel dialkylphenyl compounds having β₂adrenergic receptor agonist and muscarinic receptor antagonist activity.This invention also relates to pharmaceutical compositions comprisingsuch compounds, processes and intermediates for preparing such compoundsand methods of using such compounds, for example, to treat pulmonarydisorders.

2. State of the Art

Pulmonary disorders, such as asthma and chronic obstructive pulmonarydisease (COPD), are commonly treated with bronchodilators. One type ofbronchodilator used to treat pulmonary disorders consists of β₂adrenergic receptor (adrenoceptor) agonists, such as albuterol,formoterol and salmeterol. These compounds are generally administered byinhalation. Another type of bronchodilator consists of muscarinicreceptor antagonists (anticholinergic compounds), such as ipratropiumand tiotropium. These compounds are also typically administered byinhalation.

Pharmaceutical compositions containing a combination of a β₂ adrenergicreceptor agonist and a muscarinic receptor antagonist are known in theart for use in treating pulmonary disorders. For example, U.S. Pat. No.6,433,027, issued Aug. 13, 2002, discloses medicament compositionscontaining a muscarinic receptor antagonist, such as tiotropium bromide,and a β₂ adrenergic receptor agonist, such as formoterol fumarate.

Additionally, compounds having both β₂ adrenergic receptor agonist andmuscarinic receptor antagonist activity are known in the art. Forexample, U.S. Pat. No. 7,141,671, issued Nov. 28, 2006, disclosesbiphenyl compounds having both β₂ adrenergic receptor agonist andmuscarinic receptor antagonist activity. Compounds possessing both β₂adrenergic receptor agonist and muscarinic receptor antagonist activityare highly desirable since such compounds provide bronchodilationthrough two independent modes of action while having single moleculepharmacokinetics.

When treating pulmonary disorders, it is particularly useful to providetherapeutic agents that have a long duration of action, i.e., a durationof at least about 24 hours, when administered by inhalation so thatpatients only need to administer the therapeutic agent once a day orless. Not all dual-acting compounds disclosed previously in the artpossess this desirable property.

Accordingly, a need exists for novel compounds having both β₂ adrenergicreceptor agonist and muscarinic receptor antagonist activity thatpossess a long duration of action when administered to a patient byinhalation.

SUMMARY OF THE INVENTION

The present invention provides novel dialkylphenyl compounds having bothβ₂ adrenergic receptor agonist and muscarinic receptor antagonistactivity. Among other properties, a compound of this invention has beenfound to possess a long duration of action, i.e., a duration of at leastabout 24 hours, when administered to a mammal by inhalation.Accordingly, the compounds of this invention are expected to be usefuland advantageous as therapeutic agents for treating pulmonary disorders.

Accordingly, in one of its composition aspects, the present inventionrelates to a compound of formula I:

wherein

R¹ is methyl or ethyl; R² is methyl or ethyl; or a pharmaceuticallyacceptable salt or solvate or stereoisomer thereof.

In a particular aspect of this invention, the compound of formula I is acompound having formula II:

wherein R¹ and R² are as defined herein (including any specific orpreferred embodiments); or a pharmaceutically acceptable salt or solvatethereof.

In another of its composition aspects, this invention relates to apharmaceutical composition comprising a pharmaceutically acceptablecarrier and a compound of formula I.

If desired, the compounds of the present invention can be administeredin combination with other therapeutic agents, such as a steroidalanti-inflammatory agent. Accordingly, in another of its compositionaspects, this invention relates to a pharmaceutical compositioncomprising (a) a compound of formula I; and (b) a second therapeuticagent. In yet another of its composition aspects, this invention relatesto pharmaceutical composition comprising (a) a compound of formula I;(b) a second therapeutic agent; and (c) a pharmaceutically acceptablecarrier.

In still another of its composition aspects, this invention relates to acombination of therapeutic agents, the combination comprising (a) acompound of formula I; and (b) a second therapeutic agent. In another ofits composition aspects, this invention relates to a combination ofpharmaceutical compositions, the combination comprising (a) a firstpharmaceutical composition comprising a compound of formula I and afirst pharmaceutically acceptable carrier; and (b) a secondpharmaceutical composition comprising a second therapeutic agent and asecond pharmaceutically acceptable carrier. This invention also relatesto a kit containing such pharmaceutical compositions.

Compounds of this invention possess both β₂ adrenergic receptor agonistactivity and muscarinic receptor antagonist activity. Accordingly, thecompounds of formula I are expected to be useful as therapeutic agentsfor treating pulmonary disorders, such as asthma and chronic obstructivepulmonary disease.

Accordingly, in one of its method aspects, this invention relates to amethod for treating a pulmonary disorder, the method comprisingadministering to a patient in need of treatment a therapeuticallyeffective amount of a compound of formula I. This invention also relatesto a method of treating chronic obstructive pulmonary disease or asthma,the method comprising administering to a patient a therapeuticallyeffective amount of a compound of formula I. Additionally, in another ofits method aspects, this invention relates to a method of producingbronchodilation in a mammal, the method comprising administering to amammal a bronchodilation-producing amount of a compound of formula I.This invention also relates to method of antagonizing a muscarinicreceptor and agonizing a β₂ adrenergic receptor in a mammal, the methodcomprising administering to the mammal a compound of formula I.

Since compounds of this invention possess both β₂ adrenergic receptoragonist activity and muscarinic receptor antagonist activity, suchcompounds are also useful as research tools. Accordingly, in yet anotherof its method aspects, this invention relates to a method of using acompound of formula I as a research tool, the method comprisingconducting a biological assay using a compound of formula I.

The compounds of this invention can also be used to evaluate newchemical compounds. Accordingly, in another of its method aspects, thisinvention relates to a method of evaluating a test compound in abiological assay, the method comprising: (a) conducting a biologicalassay with a test compound to provide a first assay value; (b)conducting the biological assay with a compound of formula I to providea second assay value; wherein step (a) is conducted either before, afteror concurrently with step (b); and (c) comparing the first assay valuefrom step (a) with the second assay value from step (b).

This invention also relates to processes and novel intermediates usefulfor preparing compounds of formula I. Accordingly, in another of itsmethod aspects, this invention relates to a process of preparing acompound of formula I, the process comprising deprotecting a compound offormula 6 (as defined herein) to provide a compound of formula I.

In another of its method aspects, this invention relates to a process ofpreparing a compound of formula I, the process comprising: (a) reactinga compound of formula 4 with a compound of formula 5 in the presence ofa reducing agent to provide a compound of formula 6; and (b)deprotecting the compound of formula 6 to provide a compound of formulaI; where compounds 4, 5 and 6 are as defined herein.

In a particular embodiment of this invention, the compounds of formula Iare prepared by deprotecting a compound of formula 6, wherein thehydroxyl-protecting group is a silyl group. Accordingly, in yet anotherof its method aspects, this invention relates to a process of preparinga compound of formula I, the process comprising deprotecting a compoundof formula 6a:

wherein R^(a), R^(b) and R^(c) are independently selected from C₁₋₄alkyl, phenyl, —C₁₋₄ alkyl-(phenyl), or one of R^(1a), R^(1b) and R^(1c)is —O—(C₁₋₄ alkyl); to provide a compound of formula I.

In other embodiments, the processes described herein further comprisethe step of forming a pharmaceutically acceptable salt of a compound offormula I. In other embodiments, this invention relates to the otherprocesses described herein; and to the product prepared by any of theprocesses described herein.

In a particular embodiment, this invention relates to a compound offormula III:

or a salt or stereoisomer thereof, wherein Y¹ is selected from —CHO,—CN, —CH₂OH, —CH(OR^(3a))OR^(3b), —C(O)OH, —C(O)OR^(3c), bromo and iodo,where R^(3a) and R^(3b) are selected independently from C₁₋₆ alkyl, orR^(3a) and R^(3b) are joined to form C₂₋₆ alkylene, R^(3c) is selectedfrom C₁₋₆ alkyl; and R¹ and R² are as defined herein (including anyspecific or preferred embodiments), which compounds are useful asintermediates in preparing compounds of formula I. In a particularembodiment of formula III, R¹ and R² are methyl. In another particularembodiment of formula III, Y¹ is —CHO. In still another particularembodiment of formula III, R¹ and R² are methyl and Y¹ is —CHO.

This invention also relates to the use of a compound of formula I fortherapy. Additionally, the invention relates to the use of a compound offormula I for the manufacture of a medicament for the treatment of apulmonary disorder; and to the use of a compound of formula I as aresearch tool. Other aspects and embodiments of this invention aredisclosed herein.

DETAILED DESCRIPTION OF THE INVENTION

In one of its composition aspects, this invention relates to novelcompounds of formula I. The compounds of formula I contain one or morechiral centers and therefore, this invention is directed to racemicmixtures; pure stereoisomers (i.e., enantiomers or diastereomers);stereoisomer-enriched mixtures and the like unless otherwise indicated.When a particular stereoisomer is shown or named herein, it will beunderstood by those skilled in the art that minor amounts of otherstereoisomers may be present in the compositions of this inventionunless otherwise indicated, provided that the utility of the compositionas a whole is not eliminated by the presence of such other isomers.

In particular, compounds of formula I contain a chiral center at thecarbon atom indicated by the symbol * in the following partial formula:

In a particular embodiment of this invention, the carbon atom identifiedby the symbol * has the (R) configuration. In this embodiment, thecompounds of formula I have the (R) configuration at the carbon atomidentified by the symbol * or are enriched in a stereoisomeric formhaving the (R) configuration at this carbon atom.

The compounds of formula I also contain several basic groups (e.g.,amino groups) and therefore, the compounds of formula I can exist as thefree base or in various salt forms. All such forms are included withinthe scope of this invention. Furthermore, solvates of compounds offormula I or salts thereof are included within the scope of thisinvention.

Accordingly, those skilled in the art will recognize that reference to acompound herein, for example, reference to a compound of formula I orcompound 6, includes reference to salts and stereoisomers and solvatesof that compound unless otherwise indicated.

Additionally, as used herein, the singular forms “a,” “an” and “the”include the corresponding plural forms unless the context of use clearlydictates otherwise.

The nomenclature used herein to name the compounds of this invention andintermediates thereof has generally been derived using the commerciallyavailable AutoNom software (MDL, San Leandro, Calif.). Typically,compounds of formula I have been named as piperidin-4-yl esterderivatives of biphenyl-2-yl carbamic acid.

Representative Embodiments

The following substituents and values are intended to providerepresentative examples of various aspects and embodiments of thisinvention. These representative values are intended to further defineand illustrate such aspects and embodiments and are not intended toexclude other embodiments or to limit the scope of this invention. Inthis regard, the representation that a particular value or substituentis preferred is not intended in any way to exclude other values orsubstituents from this invention unless specifically indicated.

In one embodiment, R¹ is methyl and R² is methyl.

In another embodiment, R¹ is ethyl and R² is ethyl.

In another embodiment, R¹ is methyl and R² is ethyl.

In another embodiment, R¹ is ethyl and R² is methyl.

Thus, in one of its composition aspects, the present invention relatesto compounds of formula I selected from: biphenyl-2-ylcarbamic acid1-[2-(4-{[(R)-2-(3-formylamino-4-hydroxyphenyl)-2-hydroxyethylamino]methyl}-2,5-dimethylphenylcarbamoyl)ethyl]piperidin-4-ylester (compound IIa);

biphenyl-2-ylcarbamic acid1-[2-(4-{[(R)-2-(3-formylamino-4-hydroxyphenyl)-2-hydroxyethylamino]methyl}-2,5-diethylphenylcarbamoyl)ethyl]piperidin-4-ylester (compound IIb);

biphenyl-2-ylcarbamic acid1-[2-(4-{[(R)-2-(3-formylamino-4-hydroxyphenyl)-2-hydroxyethylamino]methyl}-2-methyl-5-ethylphenylcarbamoyl)ethyl]piperidin-4-ylester (compound IIc);

biphenyl-2-ylcarbamic acid1-[2-(4-{[(R)-2-(3-formylamino-4-hydroxyphenyl)-2-hydroxyethylamino]methyl}-2-ethyl-5-methylphenylcarbamoyl)ethyl]piperidin-4-ylester (compound IId);

or a pharmaceutically acceptable salt thereof.

DEFINITIONS

When describing the compounds, compositions, methods and processes ofthis invention, the following terms have the following meanings unlessotherwise indicated.

The term “alkyl” means a monovalent saturated hydrocarbon group whichmay be linear or branched. Unless otherwise defined, such alkyl groupstypically contain from 1 to 10 carbon atoms. Representative alkyl groupsinclude, by way of example, methyl, ethyl, n-propyl, isopropyl, n-butyl,sec-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl,n-nonyl, n-decyl and the like.

When a specific number of carbon atoms is intended for a particular termused herein, the number of carbon atoms is shown preceding the term. Forexample, the term “C₁₋₃ alkyl” means an alkyl group having from 1 to 3carbon atoms wherein the carbon atoms are in any chemically-acceptableconfiguration.

The term “alkylene” means a divalent saturated hydrocarbon group thatmay be linear or branched. Unless otherwise defined, such alkylenegroups typically contain from 1 to 10 carbon atoms. Representativealkylene groups include, by way of example, methylene, ethane-1,2-diyl(“ethylene”), propane-1,2-diyl, propane-1,3-diyl, butane-1,4-diyl,pentane-1,5-diyl and the like.

The term “amino-protecting group” means a protecting group suitable forpreventing undesired reactions at an amino group. Representativeamino-protecting groups include, but are not limited to,tert-butoxycarbonyl (BOC), trityl (Tr), benzyloxycarbonyl (Cbz),9-fluorenylmethoxycarbonyl (Fmoc), benzyl, formyl, trimethylsilyl (TMS),tert-butyldimethylsilyl (TBS), and the like.

The term “carboxy-protecting group” means a protecting group suitablefor preventing undesired reactions at a carboxy group. Representativecarboxy-protecting groups include, but are not limited to, esters, suchas methyl, ethyl, tert-butyl, benzyl (Bn), p-methoxybenzyl (PMB),9-fluorenylmethyl (Fm), trimethylsilyl (TMS), tert-butyldimethylsilyl(TBS), diphenylmethyl (benzhydryl, DPM) and the like.

The term “compound of the invention” or “compound of formula I” or“compound of formula II” as used herein means the specified compound(s)or a pharmaceutically acceptable salt or solvate or stereoisomer thereofunless otherwise indicated.

The term “halo” means fluoro, chloro, bromo and iodo.

The term “hydroxyl-protecting group” means a protecting group suitablefor preventing undesirable reactions at a hydroxyl group. Representativehydroxyl-protecting groups include, but are not limited to, silyl groupsincluding tri(C₁₋₆ alkyl)silyl groups, such as trimethylsilyl (TMS),triethylsilyl (TES), tert-butyldimethylsilyl (TBS) and the like; esters(acyl groups) including C₁₋₆ alkanoyl groups, such as formyl, acetyl andthe like; arylmethyl groups, such as benzyl (Bn), p-methoxybenzyl (PMB),9-fluorenylmethyl (Fm), diphenylmethyl (benzhydryl, DPM) and the like.Additionally, two hydroxyl groups can also be protected as an alkylidenegroup, such as prop-2-ylidine, formed, for example, by reaction with aketone, such as acetone.

The term “leaving group” means a functional group or an atom that can bedisplaced by another functional group or atom in a substitutionreaction, such as a nucleophilic substitution reaction. By way ofexample, representative leaving groups include, but are not limited to,chloro, bromo and iodo groups; sulfonic ester groups, such as mesylate,tosylate, brosylate, nosylate and the like; and acyloxy groups, such asacetoxy, trifluoroacetoxy and the like.

The term “mass median diameter” or “MMD” when used to refer to particlesmeans the diameter such that half the mass of the particles is containedin particles with larger diameter and half is contained in particleswith smaller diameter.

The term “micronized” or “in micronized form” means particles in whichat least about 90 percent of the particles have a diameter of less thanabout 10 μm unless otherwise indicated.

The term “or a pharmaceutically acceptable salt or solvate orstereoisomer thereof” as used herein is intended to include allpermutations of salts, solvates and stereoisomers, such as a solvate ofa pharmaceutically acceptable salt of a stereoisomer of a compound offormula I.

The term “pharmaceutically acceptable salt” means a salt that isacceptable for administration to a patient, such as a mammal (e.g.,salts having acceptable mammalian safety for a given dosage regime).Representative pharmaceutically acceptable salts include salts ofacetic, ascorbic, benzenesulfonic, benzoic, camphosulfonic, citric,ethanesulfonic, edisylic, fumaric, gentisic, gluconic, glucoronic,glutamic, hippuric, hydrobromic, hydrochloric, isethionic, lactic,lactobionic, maleic, malic, mandelic, methanesulfonic, mucic,naphthalenesulfonic, naphthalene-1,5-disulfonic,naphthalene-2,6-disulfonic, nicotinic, nitric, orotic, pamoic,pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonicand xinafoic acid, and the like.

The term “protected derivatives thereof” means a derivative of thespecified compound in which one or more functional groups of thecompound are protected or blocked from undergoing undesired reactionswith a protecting or blocking group. Functional groups that may beprotected include, by way of example, carboxy groups, amino groups,hydroxyl groups, thiol groups, carbonyl groups and the like. Suitableprotecting groups for such functional groups are well known to those ofordinary skill in the art as exemplified by the teachings in T. W.Greene and G. M. Wuts, Protecting Groups in Organic Synthesis, ThirdEdition, Wiley, New York, 1999, and references cited therein.

The term “salt thereof” means a compound formed when the hydrogen of anacid is replaced by a cation, such as a metal cation or an organiccation and the like. In the present invention, the cation typicallycomprises a protonated form of a compound of formula I, i.e. where oneor more amino groups having been protonated by an acid. Preferably, thesalt is a pharmaceutically acceptable salt, although this is notrequired for salts of intermediate compounds that are not intended foradministration to a patient.

The term “solvate” means a complex or aggregate formed by one or moremolecules of a solute, i.e. a compound of formula I or apharmaceutically acceptable salt thereof, and one or more molecules of asolvent. Such solvates are typically crystalline solids having asubstantially fixed molar ratio of solute and solvent. Representativesolvents include, by way of example, water, methanol, ethanol,isopropanol, acetic acid and the like. When the solvent is water, thesolvate formed is a hydrate.

The term “therapeutically effective amount” means an amount sufficientto effect treatment when administered to a patient in need of treatment.

The term “treating” or “treatment” as used herein means the treating ortreatment of a disease or medical condition (such as COPD or asthma) ina patient, such as a mammal (particularly a human) that includes:

-   -   (a) preventing the disease or medical condition from occurring,        i.e., prophylactic treatment of a patient;    -   (b) ameliorating the disease or medical condition, i.e.,        eliminating or causing regression of the disease or medical        condition in a patient;    -   (c) suppressing the disease or medical condition, i.e., slowing        or arresting the development of the disease or medical condition        in a patient; or    -   (d) alleviating the symptoms of the disease or medical condition        in a patient.

All other terms used herein are intended to have their ordinary meaningas understood by those of ordinary skill in the art to which theypertain.

General Synthetic Procedures

The compounds of this invention can be prepared from readily availablestarting materials using the following general methods and procedures orby using other information readily available to or known by those ofordinary skill in the art. Although the following procedures mayillustrate a particular embodiment or aspect of the present invention,those skilled in the art will recognize that other embodiments oraspects of the present invention can be prepared using the same orsimilar methods or by using other methods, reagents and startingmaterials known to those skilled in the art. It will also be appreciatedthat where typical or preferred process conditions (i.e., reactiontemperatures, times, mole ratios of reactants, solvents, pressures,etc.) are given, other process conditions can also be used unlessotherwise stated. While optimum reaction conditions will typically varydepending on various reaction parameters such as the reactants, solventsand quantities used, those of ordinary skill in the art can readilydetermine suitable reaction conditions using routine optimizationprocedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary or desired to preventcertain functional groups from undergoing undesired reactions. Thechoice of a suitable protecting group for a particular functional groupas well as suitable conditions and reagents for protection anddeprotection of such functional groups are well known in the art.Protecting groups other than those illustrated in the proceduresdescribed herein may be used, if desired.

In one embodiment, the compounds of formula I are synthesized asillustrated in Scheme I:

wherein P¹ is a hydroxyl-protecting group; and R^(3a) and R^(3b) areselected independently from C₁₋₆ alkyl, or R^(3a) and R^(3b) are joinedto form C₂₋₆ alkylene.

As shown in Scheme I, compound 1 can be reacted with about 0.95 to about1.05 molar equivalents of compound 2 to provide compound 3. This Michaelreaction is typically conducted at a temperature ranging from about 0°C. to about 75° C., typically about 40° C. to about 45° C., for about 8to about 24 hours or until the reaction is substantially complete.Generally, this reaction is conducted in a suitable diluent, such asdichloromethane or mixtures of dichloromethane and methanol or ethanol,and the like. Upon completion of the reaction, the product is typicallyisolated using conventional procedures, such as extraction,recrystallization, chromatography and the like. Alternatively, thereaction mixture containing compound 3 can be used directly in the nextstep of the synthesis.

Compound 3 is then reacted with aqueous acid to hydrolyze the acetalgroup and provide aldehyde compound 4. Any suitable acid can be employedin this reaction including, by way of example, hydrochloric acid,sulfuric acid, methanesulfonic acid, p-toluenesulfonic acid and thelike. The hydrolysis reaction is typically conducted at a temperatureranging from about 0° C. to about 30° C., typically about 20° C. toabout 25° C., for about 1 to about 6 hours or until the reaction issubstantially complete. Generally, this reaction is conducted in asuitable diluent, such as methanol, ethanol, isopropanol,dichloromethane/ethanol, acetonitrile and the like. Upon completion ofthe reaction, the product is typically isolated using conventionalprocedures, such as extraction, recrystallization, chromatography andthe like.

Compound 4 is then reacted with about 0.95 to about 1.5 molarequivalents of compound 5 in the presence of a reducing agent to affordcompound 6. Any suitable reducing agent may be used in this reactionincluding, by way of illustration, a metal hydride reagent, such assodium borohydride, sodium triacetoxyborohydride, sodiumcyanoborohydride and the like, or hydrogen and a metal catalyst, such aspalladium on carbon, and the like. This reductive alkylation reaction istypically conducted at a temperature ranging from about −20° C. to about30° C., typically about 0° C. to about 5° C., for about 1 to about 6hours or until the reaction is substantially complete. Generally, thisreaction is conducted in a suitable diluent and a protic solvent, suchas dichloroethane and methanol, and the like. Upon completion of thereaction, the product is typically isolated using conventionalprocedures, such as extraction, recrystallization, chromatography andthe like.

Compound 6 is then deprotected to provide a compound of formula I. Theparticular conditions used to deprotect compound 6 will depend on theprotecting group employed. For example, when P¹ is a silyl protectinggroup (i.e., a compound of formula 6a as defined herein), such astert-butyldimethylsilyl, tert-butyldiphenylsilyl, diphenylmethylsilyl,di-tert-buylmethylsilyl, tert-butoxydiphenylsilyl and the like, thisdeprotection reaction is typically conducted by contacting compound 6awith a source of fluoride ion. In a particular embodiment, the source offluoride ion is triethylamine trihydrofluoride. Other suitable sourcesof fluoride ion include tetrabutylammonium fluoride, potassium fluoridewith 18-crown-6, hydrogen fluoride, pyridine hydrofluoride, and thelike. This reaction is typically conducted at a temperature ranging fromabout 0° C. to about 50° C., typically about 10° C. to about 25° C., forabout 24 to about 72 hours or until the reaction is substantiallycomplete. Generally, this reaction is conducted in a suitable diluent,such as dichloroethane and the like. Upon completion of the reaction,the product is typically isolated using conventional procedures, such asextraction, recrystallization, chromatography and the like.

Compound 1 is known in the art or can be prepared from commerciallyavailable starting materials and reagents using known procedures. See,for example, U.S. Patent Application Publication No. U.S. 2004/0167167A1 and R. Naito et al., Chem. Pharm. Bull., 46(8) 1286-1294 (1998). Byway of example, compound I can be prepared as illustrated in Scheme II:

As shown in Scheme II, biphenyl-2-isocyanate (7) is reacted with anN-protected 4-hydroxypiperidine 8, where P² is an amino-protecting groupsuch as benzyl, to provide carbamate intermediate 9. This reaction istypically conducted at a temperature ranging from about 20° C. to about100° C., typically about 60° C. to about 80° C., for about 6 to about 24hours or until the reaction is substantially complete. If desired, thisreaction can be conducted in a suitable diluent, such asdichloromethane, toluene and the like. Alternatively, this reaction canbe conducted in the absence of a diluent. Upon completion of thereaction, the product 9 is typically isolated using conventionalprocedures, such as extraction, recrystallization, chromatography andthe like. Alternatively, the reaction mixture containing compound 2 isused directly in the next step of the synthesis.

The amino-protecting group, P², is then removed from compound 2 usingconventional procedures to afford compound 1. For example, when P² is abenzyl group, compound 2 can be deprotected using hydrogen or ammoniumformate, in the presence of a catalyst, such as a palladium catalyst.Representative catalysts include, by way of illustration, palladium oncarbon, palladium hydroxide on carbon and the like. This reaction istypically conducted at a temperature ranging from about 20° C. to about50° C., typically about 40° C., for about 6 to about 24 hours or untilthe reaction is substantially complete. Generally, this reaction isconducted in a suitable diluent, such as methanol, ethanol, isopropanoland the like. Upon completion of the reaction, compound 1 is typicallyisolated using conventional procedures, such as extraction,recrystallization, chromatography and the like.

Compounds of formula 2 are known in the art or can be prepared fromcommercially available starting materials and reagents using knownprocedures. By way of illustration, compounds of formula 2 can beprepared as shown in Scheme III:

As shown in Scheme III, an aniline compound of formula 10, where X¹ isbromo or iodo, is first protected at the amino group to provide acompound of formula 11, where P^(3a) is an amino-protecting group andP^(3b) is hydrogen or an amino-protecting group. Any suitableamino-protecting group may be used, such as benzyl, 4-methoxybenzyl,trifluoroacetyl and the like. For example, a compound of formula 10 canbe reacted with about 2 or more molar equivalents, preferably about 2.5to about 3.0 molar equivalents, of a benzyl halide, such as benzylchloride, bromide or iodide, to afford compound 11 where P^(3a) andP^(3b) are both benzyl. This reaction is typically conducted at atemperature ranging from about 0° C. to about 50° C., typically about30° C., for about 18 to about 24 hours or until the reaction issubstantially complete. Generally, this reaction is conducted in asuitable diluent, such as methanol, ethanol, isopropanol and the like.Typically, this reaction is also conducted in the presence of a suitablebase, such as potassium carbonate, sodium carbonate and the like. Uponcompletion of the reaction, compound 11 is typically isolated usingconventional procedures, such as extraction, recrystallization,chromatography and the like.

Representative compounds of formula 10 that can be employed in thisreaction include 2,5-dimethyl-4-iodoaniline, 2,5-diethyl-4-iodoaniline,2-ethyl-4-iodo-5-methylaniline, 5-ethyl-4-iodo-2-methylaniline,4-bromo-2,5-dimethylaniline, 4-bromo-2,5-diethylaniline,4-bromo-2-ethyl-5-methylaniline, 4-bromo-5-ethyl-2-methylaniline and thelike. Such compounds are commercially available (for example, fromSpectra Group Limited, Inc., Millbury, Ohio) or can be prepared fromcommercially available starting materials and reagents usingconventional procedures.

Compound 11 is then contacted with about 1 to about 2 molar equivalentsof an alkyl lithium reagent, such as n-butyllithium ortert-butyllithium, to form the corresponding anion in which the X¹ grouphas been exchanged for lithium. This reaction is typically conducted ata temperature ranging from about −70° C. to about 0° C., typically about−20° C., for about 0.25 to about 1 hour or until the reaction issubstantially complete. Generally, this reaction is conducted in asuitable diluent, such as toluene, xylene, tetrahydrofuran and the like.

The resulting lithium anion is not isolated, but is reacted in situ witha molar excess of N,N-dimethylformamide to provide compound 12.Generally, about 2 to about 4 molar equivalents of N,N-dimethylformamideare used. This reaction is typically conducted at a temperature rangingfrom about −70° C. to about 0° C., typically about −20° C. to about 0°C., for about 0.5 to about 2 hours or until the reaction issubstantially complete. Upon completion of the reaction, compound 12 istypically isolated using conventional procedures, such as extraction,recrystallization, chromatography and the like.

The aldehyde group of compound 12 is then protected as an acetal byreacting compound 12 with an alcohol or a diol in the presence of anacid catalyst. Any suitable alcohol or diol can be used in thisreaction. For example, representative alcohols and diols includemethanol, ethanol, n-propanol, ethylene glycol, propylene glycol and thelike. Generally, a molar excess of the alcohol or diol are employed inthis reaction, preferably about 2 to about 4 molar equivalents.

Any suitable acid catalyst may be used in this reaction to facilitateformation of the acetal. Representative acid catalysis include, by wayof example, p-toluenesulfonic acid, benzenesulfonic acid, hydrochloricacid and the like.

The reaction is typically conducted at a temperature ranging from about50° C. to about 100° C., typically about 60° C. to about 80° C., forabout 12 to about 24 hours or until the reaction is substantiallycomplete. Generally, this reaction is conducted in a suitable diluent,such as toluene, xylene and the like. Typically, the reaction isconducted in a manner which allows the water produced to be removed,such as by azeotropic distillation or by the use of molecular sieves.Upon completion of the reaction, compound 13 is typically isolated usingconventional procedures, such as extraction, recrystallization,chromatography and the like. Alternatively, the reaction mixturecontaining compound 13 can be used directly in the next step of thesynthesis.

After formation of the acetal, the amino group of compound 13 isdeprotected using standard reagents and conditions to form compound 14.For example, if P^(3a) and P^(3b) are benzyl groups, compound 13 can bedeprotected using hydrogen and a catalyst, such as a palladium catalyst.Representative catalysts include, by way of example, palladium oncarbon, palladium hydroxide on carbon, and the like. This reaction istypically conducted at a temperature ranging from about 20° C. to about50° C., typically about 25° C. to about 30° C., for about 4 to about 12hours or until the reaction is substantially complete. Generally, thisreaction is conducted in a suitable diluent, such as methanol, ethanol,ethanol/ethyl acetate mixtures and the like. Upon completion of thereaction, compound 14 is typically isolated using conventionalprocedures, such as extraction, recrystallization, chromatography andthe like.

Compound 14 is then reacted with an acryloyl halide 15, where X² ischloro, bromo or iodo, to form compound 2. This reaction is typicallyconducted at a temperature ranging from about −20° C. to about 25° C.,typically about 0° C. to about 5° C., for about 0.5 to about 6 hours oruntil the reaction is substantially complete. Generally, this reactionis conducted in a suitable diluent, such as dichloromethane and thelike, in the presence of a suitable base, such as diisopropylethylamine,triethylamine and the like. Generally, about 1 to 1.2 molar equivalentsof the acryloyl halide and about 1 to about 2 molar equivalent of thebase are used in this reaction. Upon completion of the reaction,compound 2 is typically isolated using conventional procedures, such asextraction, recrystallization, chromatography and the like.

Compounds of formula 5 are known in the art or can be prepared fromcommercially available starting materials and reagents using knownprocedures. By way of illustration, compounds of formula 5 can beprepared as shown in Scheme IV:

As illustrated in Scheme IV, compounds of formula 5 can be prepared fromcompounds of formula 16, where P⁴ is a hydroxyl-protecting group, suchas benzyl, and X³ is a leaving group, such as chloro, bromo or iodo.Compounds of formula 16 are known in the art. For example, U.S. Pat. No.6,268,533 B1, issued Jul. 31, 2001; and R. Hett et al., Organic ProcessResearch & Development 1998, 2, 96-99; describe the preparation ofN-[2-benzyloxy-5-((R)-2-bromo-1-hydroxyethyl)phenyl]formamide (i.e., the(R) enantiomer of compound 16, where P⁴ is benzyl and X³ is bromo)starting from 2-bromo-4′-benzyloxy-3′-nitroacetophenone, the synthesisof which is described in K. Murase et al., Chem. Pharm. Bull., 25(6)1368-1377 (1977). If desired, the racemic form of compound 16 can beprepared by using a non-chiral reducing agent, such as boranedimethylsulfide complex, to reduce2-bromo-4′-benzyloxy-3′-nitroacetophenone.

The hydroxyl group of compound 16 is protected using conventionalprocedures and reagents to provide compound 17, where P¹ is ahydroxyl-protecting group. In a particular embodiment, thehydroxyl-protecting group is a silyl protecting group, such asdimethylisopropylsilyl, diethylisopropylsilyl, dimethylhexylsilyl,tert-butyldimethylsilyl, tert-butyldiphenylsilyl, diphenylmethylsilyland the like. For example, compound 16 can be reacted with about 0.95 toabout 1.2 molar equivalent of tert-butyldimethylsilyl chloride in thepresence of about 1.1 to about 1.3 molar equivalents of imidazole toprovide compound 17 where P¹ is tert-butyldimethylsilyl. This reactionis typically conducted at a temperature ranging from about 0° C. toabout 50° C., typically at room temperature, for about 24 to about 48hours or until the reaction is substantially complete. Generally, thisreaction is conducted in a suitable diluent, such asN,N-dimethylformamide and the like. Upon completion of the reaction,compound 17 is typically isolated using conventional procedures, such asextraction, chromatography and the like. By way of further illustration,the synthesis ofN-{2-benzyloxy-5-[(R)-2-bromo-1-(tert-butyldimethylsilanyloxy)ethyl]phenyl}formamideis described in Example 2 of U.S. Patent Publication No. US 2004/0248985A1, published Dec. 9, 2004.

Compound 17 is then reacted with a benzyl amine (i.e., Bn¹—NH₂) toafford compound 18, where Bn¹ is an unsubstituted benzyl group or asubstituted benzyl group having 1 to 3 substituents on the phenyl ringof the benzyl group selected independently from C₁₋₄ alkyl or C₁₋₄alkoxy. Representative benzyl amines include, benzylamine,3,4-dimethoxybenzylamine, 4-methoxybenzylamine, 4-methylbenzylamine andthe like. This reaction is typically conducted by contacting compound 17with about 2 to about 4 molar equivalents of the benzyl amine at atemperature ranging from about 40° C. to about 100° C., typically fromabout 80° C. to about 90° C., for about 5 to about 24 hours or until thereaction is substantially complete. Generally, this reaction isconducted in a suitable diluent, such as N-methyl-2-pyrrolidone (NMP)and the like. Upon completion of the reaction, compound 18 is typicallyisolated using conventional procedures, such as extraction,chromatography, recrystallization and the like.

Removal of the benzyl group, Bn¹, and P⁴ using conventional proceduresand reagents then affords compound 5. In one embodiment, both Bn₁ and P⁴are benzyl groups that are removed in the same reaction mixture.Typically, this reaction is conducted by contacting compound 5 withhydrogen in the presence of a catalyst, such as a palladium catalyst.Representative catalysts include palladium hydroxide on carbon,palladium on carbon, and the like. Generally, this debenzylationreaction is conducted in the presence of an acid, such as acetic acid,formic acid and the like. This reaction is typically conducted at atemperature ranging from about 10° C. to about 50° C., typically at roomtemperature, for about 6 to about 24 hours or until the reaction issubstantially complete. Generally, this reaction is conducted in asuitable diluent, such as methanol, ethanol and the like. Uponcompletion of the reaction, compound 5 is typically isolated usingconventional procedures, such as extraction, chromatography and thelike. In a particular embodiment, compound 5 is isolated as the aceticacid salt.

Those of ordinary skill in the art will recognize that the compounds offormula I can also be prepared by other synthetic procedures. Forexample, the particular order in which the synthetic steps are conductedcan be changed or different intermediates can be employed. By way ofillustration, compounds of formula III:

where Y¹ is —CN, —C(O)OH or —C(O)OR^(3c) can be reduced usingconventional procedures and reagents to provide aldehyde 4 (i.e., whereY¹ is —CHO). Additionally, such compounds can be reduced to the alcohol,i.e., where Y¹ is —CH₂OH, and the alcohol can then be oxidized usingstandard procedures and reagents to provide aldehyde 4 (i.e., where Y¹is —CHO).

If desired, pharmaceutically acceptable salts of the compounds offormula I can be prepared by contacting the free base form of a compoundof formula I with a pharmaceutically acceptable acid.

Further details regarding specific reaction conditions and otherprocedures for preparing representative compounds of this invention orintermediates thereof are described in the Examples set forth below.

Pharmaceutical Compositions Combinations and Formulations

The compounds of this invention are typically administered to a patientin the form of a pharmaceutical composition or formulation. Suchpharmaceutical compositions may be administered to the patient by anyacceptable route of administration including, but not limited to,inhaled, oral, nasal, topical (including transdermal) and parenteralmodes of administration. It will be understood that any form of acompound of this invention, (i.e., free base, pharmaceuticallyacceptable salt, solvate, etc.) that is suitable for a particular modeof administration can be used in the pharmaceutical compositionsdiscussed herein.

Accordingly, in one of its compositions aspects, this invention relatesto a pharmaceutical composition comprising a pharmaceutically acceptablecarrier or excipient and a compound of formula I. Optionally, suchpharmaceutical compositions may contain other therapeutic and/orformulating agents if desired.

The pharmaceutical compositions of this invention typically contain atherapeutically effective amount of a compound of formula I. Thoseskilled in the art will recognize, however, that a pharmaceuticalcomposition may contain more than a therapeutically effective amount,i.e., bulk compositions, or less than a therapeutically effectiveamount, i.e., individual unit doses designed for multiple administrationto achieve a therapeutically effective amount.

Typically, the pharmaceutical composition will contain from about 0.01to about 95 percent by weight of the therapeutic agent; including, fromabout 0.01 to about 30 percent by weight; such as from about 0.01 toabout 10 percent by weight of the therapeutic agent.

Any conventional carrier or excipient may be used in the pharmaceuticalcompositions of this invention. The choice of a particular carrier orexcipient, or combinations of carriers or excipients, will depend on themode of administration being used to treat a particular patient or typeof medical condition or disease state. In this regard, the preparationof a suitable pharmaceutical composition for a particular mode ofadministration is well within the scope of those of ordinary skill inthe pharmaceutical arts.

The carriers or excipients used in the pharmaceutical compositions ofthis invention are commercially available. For example, such materialscan be purchased from Sigma (St. Louis, Mo.). By way of furtherillustration, conventional formulation techniques are well known tothose of ordinary skill in the art as exemplified by the teachings inRemington: The Science and Practice of Pharmacy, 20^(th) Edition,Lippincott Williams & White, Baltimore, Md. (2000); and H. C. Ansel etal., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7^(th)Edition, Lippincott Williams & White, Baltimore, Md. (1999).

Representative examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, the following: (1)sugars, such as lactose, glucose and sucrose; (2) starches, such as cornstarch and potato starch; (3) cellulose, and its derivatives, such assodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;(4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8)excipients, such as cocoa butter and suppository waxes; (9) oils, suchas peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,corn oil and soybean oil; (10) glycols, such as propylene glycol; (11)polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;(12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18)Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions;(21) compressed propellant gases, such as chlorofluorocarbons andhydrofluorocarbons; and (22) other non-toxic compatible substancesemployed in pharmaceutical compositions.

The pharmaceutical compositions of this invention are typically preparedby thoroughly and intimately mixing or blending a compound of theinvention with a pharmaceutically acceptable carrier and any optionalingredients. If necessary or desired, the resulting uniformly blendedmixture can then be shaped or loaded into tablets, capsules, pills,canisters, cartridges, dispensers and the like using conventionalprocedures and equipment.

In one embodiment, the pharmaceutical compositions of this invention aresuitable for inhaled administration. Suitable pharmaceuticalcompositions for inhaled administration will typically be in the form ofan aerosol or a powder. Such compositions are generally administeredusing well-known delivery devices, such as a nebulizer inhaler, ametered-dose inhaler (MDI), a dry powder inhaler (DPI) or a similardelivery device.

In a specific embodiment of this invention, the pharmaceuticalcomposition comprising the therapeutic agent is administered byinhalation using a nebulizer inhaler. Such nebulizer devices typicallyproduce a stream of high velocity air that causes the pharmaceuticalcomposition comprising the therapeutic agent to spray as a mist that iscarried into the patient's respiratory tract. Accordingly, whenformulated for use in a nebulizer inhaler, the therapeutic agent istypically dissolved in a suitable carrier to form a solution.Alternatively, the therapeutic agent can be micronized and combined witha suitable carrier to form a suspension of micronized particles.Nebulizer devices suitable for administering therapeutic agents byinhalation are well known to those of ordinary skill in the art or suchdevices are commercially available. For example, representativenebulizer devices or products include the Respimat Softmist Inhalaler(Boehringer Ingelheim); the AERxPulmonary Delivery System (AradigmCorp.); the PARI LC Plus Reusable Nebulizer (Pari GmbH); and the like.

A representative pharmaceutical composition for use in a nebulizerinhaler comprises an isotonic aqueous solution comprising from about0.05 μg/mL to about 10 mg/mL of a compound of formula I. In oneembodiment, such a solution has a pH of about 4 to about 6.

In another specific embodiment of this invention, the pharmaceuticalcomposition comprising the therapeutic agent is administered byinhalation using a dry powder inhaler. Such dry powder inhalerstypically administer the therapeutic agent as a free-flowing powder thatis dispersed in a patient's air-stream during inspiration. In order toachieve a free-flowing powder, the therapeutic agent is typicallyformulated with a suitable excipient such as lactose, starch, mannitol,dextrose, polylactic acid (PLA), polylactide-co-glycolide (PLGA) orcombinations thereof. Typically, the therapeutic agent is micronized andcombined with a suitable carrier to form a blend suitable forinhalation. Accordingly, in one embodiment of the invention, thecompound of formula I is in micronized form.

A representative pharmaceutical composition for use in a dry powderinhaler comprises dry milled lactose and micronized particles of acompound of formula I.

Such a dry powder formulation can be made, for example, by combining thelactose with the therapeutic agent and then dry blending the components.Alternatively, if desired, the therapeutic agent can be formulatedwithout an excipient. The pharmaceutical composition is then typicallyloaded into a dry powder dispenser, or into inhalation cartridges orcapsules for use with a dry powder delivery device.

Dry powder inhaler delivery devices suitable for administeringtherapeutic agents by inhalation are well known to those of ordinaryskill in the art or such devices are commercially available. Forexample, representative dry powder inhaler delivery devices or productsinclude Aeolizer (Novartis); Airmax (IVAX); ClickHaler (InnovataBiomed); Diskhaler (GlaxoSmithKline); Diskus/Accuhaler(GlaxoSmithKline); Easyhaler (Orion Pharma); Eclipse (Aventis); FlowCaps(Hovione); Handihaler (Boehringer Ingelheim); Pulvinal (Chiesi);Rotahaler (GlaxoSmithKline); SkyeHaler/Certihaler (SkyePharma);Twisthaler (Schering-Plough); Turbuhaler (AstraZeneca); Ultrahaler(Aventis); and the like.

In yet another specific embodiment of this invention, the pharmaceuticalcomposition comprising the therapeutic agent is administered byinhalation using a metered-dose inhaler. Such metered-dose inhalerstypically discharge a measured amount of the therapeutic agent using acompressed propellant gas. Accordingly, pharmaceutical compositionsadministered using a metered-dose inhaler typically comprise a solutionor suspension of the therapeutic agent in a liquefied propellant. Anysuitable liquefied propellant may be employed includingchlorofluorocarbons, such as CCl₃F, and hydrofluoroalkanes (HFAs), suchas 1,1,1,2-tetrafluoroethane (HFA 134a) and1,1,1,2,3,3,3-heptafluoro-n-propane, (HFA 227). Due to concerns aboutchlorofluorocarbons affecting the ozone layer, formulations containingHFAs are generally preferred. Additional optional components of HFAformulations include co-solvents, such as ethanol or pentane, andsurfactants, such as sorbitan trioleate, oleic acid, lecithin, andglycerin.

A representative pharmaceutical composition for use in a metered-doseinhaler comprises from about 0.01% to about 5% by weight of a compoundof formula I; from about 0% to about 20% by weight ethanol; and fromabout 0% to about 5% by weight surfactant; with the remainder being anHFA propellant.

Such compositions are typically prepared by adding chilled orpressurized hydrofluoroalkane to a suitable container containing thetherapeutic agent, ethanol (if present) and the surfactant (if present).To prepare a suspension, the therapeutic agent is micronized and thencombined with the propellant. The formulation is then loaded into anaerosol canister, which forms a portion of a metered-dose inhalerdevice.

Metered-dose inhaler devices suitable for administering therapeuticagents by inhalation are well known to those of ordinary skill in theart or such devices are commercially available. For example,representative metered-dose inhaler devices or products include AeroBidInhaler System (Forest Pharmaceuticals); Atrovent Inhalation Aerosol(Boehringer Ingelheim); Flovent (GlaxoSmithKline); Maxair Inhaler (3M);Proventil Inhaler (Schering); Serevent Inhalation Aerosol(GlaxoSmithKline); and the like.

In another embodiment, the pharmaceutical compositions of this inventionare suitable for oral administration. Suitable pharmaceuticalcompositions for oral administration may be in the form of capsules,tablets, pills, lozenges, cachets, dragees, powders, granules; or as asolution or a suspension in an aqueous or non-aqueous liquid; or as anoil-in-water or water-in-oil liquid emulsion; or as an elixir or syrup;and the like; each containing a predetermined amount of a compound ofthe present invention as an active ingredient.

When intended for oral administration in a solid dosage form (i.e., ascapsules, tablets, pills and the like), the pharmaceutical compositionsof this invention will typically comprise a compound of the presentinvention as the active ingredient and one or more pharmaceuticallyacceptable carriers, such as sodium citrate or dicalcium phosphate.Optionally or alternatively, such solid dosage forms may also comprise:(1) fillers or extenders, such as starches, lactose, sucrose, glucose,mannitol, and/or silicic acid; (2) binders, such ascarboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; (3) humectants, such as glycerol; (4)disintegrating agents, such as agar-agar, calcium carbonate, potato ortapioca starch, alginic acid, certain silicates, and/or sodiumcarbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as cetyl alcohol and/or glycerol monostearate; (8)absorbents, such as kaolin and/or bentonite clay; (9) lubricants, suchas talc, calcium stearate, magnesium stearate, solid polyethyleneglycols, sodium lauryl sulfate, and/or mixtures thereof; (10) coloringagents; and (11) buffering agents.

Release agents, wetting agents, coating agents, sweetening, flavoringand perfuming agents, preservatives and antioxidants can also be presentin the pharmaceutical compositions of this invention. Examples ofpharmaceutically acceptable antioxidants include: (1) water-solubleantioxidants, such as ascorbic acid, cysteine hydrochloride, sodiumbisulfate, sodium metabisulfate sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal-chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like. Coating agents fortablets, capsules, pills and like, include those used for entericcoatings, such as cellulose acetate phthalate (CAP), polyvinyl acetatephthalate (PVAP), hydroxypropyl methylcellulose phthalate, methacrylicacid!methacrylic acid ester copolymers, cellulose acetate trimellitate(CAT), carboxymethyl ethyl cellulose (CMEC), hydroxypropyl methylcellulose acetate succinate (HPMCAS), and the like.

If desired, the pharmaceutical compositions of the present invention mayalso be formulated to provide slow or controlled release of the activeingredient using, by way of example, hydroxypropyl methyl cellulose invarying proportions; or other polymer matrices, liposomes and/ormicrospheres.

In addition, the pharmaceutical compositions of the present inventionmay optionally contain opacifying agents and may be formulated so thatthey release the active ingredient only, or preferentially, in a certainportion of the gastrointestinal tract, optionally, in a delayed manner.Examples of embedding compositions which can be used include polymericsubstances and waxes. The active ingredient can also be inmicro-encapsulated form, if appropriate, with one or more of theabove-described excipients.

Suitable liquid dosage forms for oral administration include, by way ofillustration, pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. Such liquid dosage formstypically comprise the active ingredient and an inert diluent, such as,for example, water or other solvents, solubilizing agents andemulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, oils (esp., cottonseed, groundnut, corn, germ,olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol,polyethylene glycols and fatty acid esters of sorbitan, and mixturesthereof. Suspensions, in addition to the active ingredient, may containsuspending agents such as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminium metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

When intended for oral administration, the pharmaceutical compositionsof this invention may be packaged in a unit dosage form. The term “unitdosage form” means a physically discrete unit suitable for dosing apatient, i.e., each unit containing a predetermined quantity of activeagent calculated to produce the desired therapeutic effect either aloneor in combination with one or more additional units. For example, suchunit dosage forms may be capsules, tablets, pills, and the like.

The compounds of this invention can also be administered transdermallyusing known transdermal delivery systems and excipents. For example, acompound of this invention can be admixed with permeation enhancers,such as propylene glycol, polyethylene glycol monolaurate,azacycloalkan-2-ones and the like, and incorporated into a patch orsimilar delivery system. Additional excipients including gelling agents,emulsifiers and buffers, may be used in such transdermal compositions ifdesired.

Additionally, the compounds of this invention can be administeredparenterally, i.e., intravenously, subcutaneously or intramuscularly.For parenteral administration, a compound of formula I is typicallydissolved in a carrier acceptable for parenteral administration, such assterile water, saline, vegetable oil and the like. By way ofillustration, an intravenous composition typically comprises a sterileaqueous solution of a compound of formula I, wherein the solution has apH in the range of about 4 to about 7.

If desired, the compounds of this invention may be administered incombination with one or more other therapeutic agents. In thisembodiment, a compound of this invention is either physically mixed withthe other therapeutic agent to form a composition containing bothagents; or each agent is present in separate and distinct compositionswhich are administered to the patient simultaneously or sequentially.

For example, a compound of formula I can be combined with secondtherapeutic agent using conventional procedures and equipment to form acomposition comprising a compound of formula I and a second therapeuticagent. Additionally, the therapeutic agents may be combined with apharmaceutically acceptable carrier to form a pharmaceutical compositioncomprising a compound of formula I, a second therapeutic agent and apharmaceutically acceptable carrier. In this embodiment, the componentsof the composition are typically mixed or blended to create a physicalmixture. The physical mixture is then administered in a therapeuticallyeffective amount using any of the routes described herein.

Alternatively, the therapeutic agents may remain separate and distinctbefore administration to the patient. In this embodiment, thetherapeutic agents are not physically mixed together beforeadministration but are administered simultaneously or sequentially asseparate compositions. For example, a compound of formula I can beadministered by inhalation simultaneously or sequentially with anothertherapeutic agent using an inhalation delivery device that employsseparate compartments (e.g. blister packs) for each therapeutic agent.Alternatively, the combination may be administered using separatedelivery devices, i.e., one delivery device for each therapeutic agent.Additionally, the therapeutic agents can be delivered by differentroutes of administration, i.e., one by inhalation and the other by oraladministration.

Any therapeutic agent compatible with the compounds of the presentinvention may be used in combination with such compounds. In aparticular embodiment, the second therapeutic agent is one that iseffectively administered by inhalation. By way of illustration,representative types of therapeutic agents that may be used with thecompounds of this invention include, but are not limited to,anti-inflammatory agents, such as steroidal anti-inflammatory agents(including corticosteroids and glucocorticoids), non-steroidalanti-inflammatory agents (NSAIDs), and PDE₄ inhibitors; bronchodilators,such as PDE₃ inhibitors, adenosine 2b modulators and β₂ adrenergicreceptor agonists; antiinfective agents, such as Gram-positiveantibiotics, Gram-negative antibiotics, and antiviral agents;antihistamines; protease inhibitors; afferent blockers, such as D₂agonists and neurokinin modulators; and muscarinic receptor antagonists(antichlolinergic agents). Numerous examples of such therapeutic agentsare well known in the art. Suitable doses for the other therapeuticagents administered in combination with a compound of the invention aretypically in the range of about 0.05 μg/day to about 500 mg/day.

In a particular embodiment of this invention, a compound of formula I isadministered in combination with a steroidal anti-inflammatory agent.Representative examples of steroidal anti-inflammatory agents that canbe used in combination with the compounds of this invention include, butare not limited to, beclomethasone dipropionate; budesonide; butixocortpropionate;20R-16α,17α-[butylidenebis(oxy)]-6α,9α-difluoro-11β-hydroxy-17β-(methylthio)androsta-4-en-3-one(RPR-106541); ciclesonide; dexamethasone;6α,9α-difluoro-17α-[(2-furanylcarbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxoandrosta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester;6α,9α-difluoro-11β-hydroxy-16α-methyl-17α-[(4-methyl-1,3-thiazole-5-carbonyl)oxy]-3-oxoandrosta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester;6α,9α-difluoro-11β-hydroxy-16α-methyl-3-oxo-17α-propionyloxyandrosta-1,4-diene-17β-carbothioicacid (S)-(2-oxotetrahydrofuran-3S-yl) ester; flunisolide; fluticasonepropionate; methyl prednisolone; mometasone furoate; prednisolone;prednisone; rofleponide; ST-126; triamcinolone acetonide; and the like,or pharmaceutically acceptable salts thereof. Such steroidalanti-inflammatory agents are commercially available or can be preparedusing conventional procedures and reagents. For example, the preparationand use of steroidal anti-inflammatory agents is described in U.S. Pat.No. 6,750,210 B2, issued Jun. 15, 2004; U.S. Pat. No. 6,759,398 B2,issued Jul. 6, 2004; U.S. Pat. No. 6,537,983, issued Mar. 25, 2003; U.S.Patent Application Publication No. US 2002/0019378 A1, published Feb.14, 2002; and references cited therein.

When employed, the steroidal anti-inflammatory agent is typicallyadministered in an amount that produces a therapeutically beneficialeffect when co-administered with a compound of the invention. Typically,the steroidal anti-inflammatory agent will be administered in an amountsufficient to provide from about 0.05 μg to about 500 μg per dose.

The following examples illustrate representative pharmaceuticalcompositions of the present invention:

Example A Dry Powder Composition

A micronized compound of the invention (100 mg) is blended with milledlactose (25 g) (e.g., lactose in which not greater than about 85% of theparticles have a MMD of about 60 μm to about 90 μm and not less than 15%of the particles have a MMD of less then 15 μm). This blended mixture isthen loaded into individual blisters of a peelable blister pack in anamount sufficient to provide about 10 μg to about 500 μg of the compoundof the invention per dose. The contents of the blisters are administeredusing a dry powder inhaler.

Example B Dry Powder Composition

A micronized compound of the invention (1 g) is blended with milledlactose (200 g) to form a bulk composition having a weight ratio ofcompound to milled lactose of 1:200. The blended composition is packedinto a dry powder inhalation device capable of delivering between about10 μg to about 500 μg of the compound of the invention per dose.

Example C Dry Powder Composition

A micronized compound of the invention (100 mg) and a micronizedsteroidal anti-inflammatory agent (500 mg) are blended with milledlactose (30 g). This blended mixture is then loaded into individualblisters of a peelable blister pack in an amount sufficient to provideabout 10 μg to about 500 μg of the compound of the invention per dose.The contents of the blisters are administered using a dry powderinhaler.

Example D Metered-Dose Inhaler Composition

A micronized compound of the invention (10 g) is dispersed in a solutionprepared by dissolving lecithin (0.2 g) in demineralized water (200 mL).The resulting suspension is spray dried and then micronized to form amicronized composition comprising particles having a mean diameter lessthan about 1.5 μm. The micronized composition is then loaded intometered-dose inhaler cartridges containing pressurized1,1,1,2-tetrafluoroethane in an amount sufficient to provide about 10 μgto about 500 μg of the compound of the invention per dose whenadministered by the metered dose inhaler.

Example E Nebulizer Composition

A compound of the invention (25 mg) is dissolved in citrate buffered (pH5) isotonic saline (125 mL). The mixture is stirred and sonicated untilthe compound is dissolved. The pH of the solution is checked andadjusted, if necessary, to pH 5 by slowly adding aqueous 1N sodiumhydroxide. The solution is administered using a nebulizer device thatprovides about 10 μg to about 500 μg of the compound of the inventionper dose.

Example F Hard Gelatin Capsules

A compound of the invention (50 g), spray-dried lactose (440 g) andmagnesium stearate (10 g) are thoroughly blended. The resultingcomposition is loaded into a hard gelatin capsule (500 mg of compositionper capsule) that are administered orally.

Example G Oral Suspension

The following ingredients are thoroughly mixed to form a suspension fororal administration:

Ingredients Amount Compound of the invention 1.0 g Fumaric acid 0.5 gSodium chloride 2.0 g Methyl paraben 0.15 g Propyl paraben 0.05 gGranulated sugar 25.5 g Sorbitol (70% solution) 12.85 g VEEGUM ® K(magnesium aluminum silicate) 1.0 g Flavoring 0.035 mL Coloring 0.5 mgDistilled water q.s. to 100 mL

The resulting suspension contains 100 mg of active ingredient per 10 mLof suspension. The suspension is administered orally.

Example H Injectable Composition

A compound of the invention (0.2 g) is blended with 0.4 M sodium acetatebuffer solution (2.0 mL). The pH of the resulting solution is adjustedto pH 4 using 0.5 N aqueous hydrochloric acid or 0.5 N aqueous sodiumhydroxide, as necessary, and then sufficient water for injection isadded to provide a total volume of 20 mL. The mixture is then filteredthrough a sterile filter (0.22 micron) to provide a sterile solutionsuitable for administration by injection.

Utility

The compounds of this invention possess both β₂ adrenergic receptoragonist and muscarinic receptor antagonist activity and therefore, suchcompounds are expected to be useful as therapeutic agents for treatingmedical conditions mediated by β₂ adrenergic receptors or muscarinicreceptors, i.e., medical conditions that are ameliorated by treatmentwith a β₂ adrenergic receptor agonist or a muscarinic receptorantagonist. Such medical conditions are well known to those of ordinaryskill in the art as exemplified by the teachings of Eglen et al.,Muscarinic Receptor Subtypes: Pharmacology and Therapuetic Potential,DN&P 10(8), 462-469 (1997); Emilien et al., Current Therapeutic Uses andPotential of beta-Adrenoceptor Agonists and Antagonists, European J.Clinical Pharm., 53(6), 389-404 (1998); and references cited therein.Such medical conditions include, by way of example, pulmonary disordersor diseases associated with reversible airway obstruction, such aschronic obstructive pulmonary disease (e.g., chronic and wheezybronchitis and emphysema), asthma, pulmonary fibrosis and the like.Other conditions include premature labor, depression, congestive heartfailure, skin diseases (e.g., inflammatory, allergic, psoriatic andproliferative skin diseases), conditions where lowering peptic acidityis desirable (e.g., peptic and gastric ulceration) and muscle wastingdisease.

Accordingly, in one embodiment, this invention relates to a method fortreating a pulmonary disorder, the method comprising administering to apatient in need of treatment a therapeutically effective amount of acompound of formula I. When used to treat a pulmonary disorder, thecompounds of this invention will typically be administered by inhalationin multiple doses per day, in a single dose per day or a single dose perweek. Generally, the dose for treating a pulmonary disorder will rangefrom about 10 μg/day to about 500 μg/day.

In one of its method aspects, this invention relates to a method oftreating chronic obstructive pulmonary disease or asthma, the methodcomprising administering to a patient a therapeutically effective amountof a compound of formula I. Generally, the dose for treating COPD orasthma will range from about 10 μg/day to about 500 μg/day. The term“COPD” is understood by those of ordinary skill in the art to include avariety of respiratory conditions, including chronic obstructivebronchitis and emphysema, as exemplified by the teachings of Barnes,Chronic Obstructive Pulmonary Disease, N. Engl. J. Med., 2000:343:269-78, and references cited therein.

When administered by inhalation, the compounds of this inventiontypically have the effect of producing bronchodilation. Accordingly, inanother of its method aspects, this invention relates to a method ofproducing bronchodilation in a mammal, the method comprisingadministering to a mammal a bronchodilation-producing amount of acompound of formula I. Generally, the dose for producing bronchodilationwill range from about 10 μg/day to about 500 μg/day.

When used as a therapeutic agent, the compounds of this invention areoptionally administered in combination with another therapeutic agent oragents. In particular, by administering the compounds of this inventionwith a steroidal anti-inflammatory agent, triple therapy, i.e., β₂adrenergic receptor agonist activity, muscarinic receptor antagonistactivity and anti-inflammatory activity, can be achieved using only twotherapeutic agents. Since pharmaceutical compositions (and combinations)containing two therapeutic agents are typically easier to formulateand/or administer compared to compositions containing three therapeuticagents, such two component compositions provide a significant advantageover compositions containing three therapeutic agents. Accordingly, in aparticular embodiment, the pharmaceutical compositions, combinations andmethods of this invention further comprise a steroidal anti-inflammatoryagent.

Since compounds of this invention possess both β₂ adrenergic agonistactivity and muscarinic receptor antagonist activity, such compounds arealso useful as research tools for investigating or studying biologicalsystems or samples having β₂ adrenergic receptors or muscarinicreceptors. Additionally, such compounds are useful in screening assaysto discover, for example, new compounds having both β₂ adrenergicagonist activity and muscarinic receptor antagonist activity. Suchbiological systems or samples may comprise β₂ adrenergic receptorsand/or muscarinic receptors. Any suitable biological system or samplehaving β₂ adrenergic and/or muscarinic receptors may be employed in suchstudies which may be conducted either in vitro or in vivo.Representative biological systems or samples suitable for such studiesinclude, but are not limited to, cells, cellular extracts, plasmamembranes, tissue samples, mammals (such as mice, rats, guinea pigs,rabbits, dogs, pigs, etc.), and the like.

When used as a research tool, a biological system or sample comprising aβ₂ adrenergic receptor and/or a muscarinic receptor is typicallycontacted with a β₂ adrenergic receptor-agonizing or muscarinicreceptor-antagonizing amount of a compound of this invention. Theeffects on the biological system or sample caused by the compound arethen determined or measured using conventional procedures and equipment,such as by measuring binding in a radioligand binding assays orligand-mediated changes in a functional assay or by determining theamount of bronchoprotection provided by the compound in abronchoprotection assay in a mammal. Representative ligand-mediatedchanges in a functional assay include ligand-mediated changes inintracellular cyclic adenosine monophosphate (cAMP); ligand-mediatedchanges in activity of the enzyme adenylyl cyclase (which synthesizescAMP); ligand-mediated changes in incorporation of guanosine5′-O-(thio)triphosphate ([³⁵S]GTP S) into isolated membranes viareceptor catalyzed exchange of [³⁵S]GTP S for GDP; ligand-mediatedchanges in free intracellular calcium ions (measured, for example, witha fluorescence-linked imaging plate reader or FLIPR® from MolecularDevices, Inc.); and the like. Compounds of this invention are expectedto agonize or cause activation of a β₂ adrenergic receptor andantagonize or decrease the activation of muscarinic receptors in thefunctional assays listed herein or in assays of a similar nature. Thecompounds of this invention will typically be used in these studies at aconcentration ranging from about 0.1 nanomolar to about 100 nanomolar.

Additionally, the compounds of this invention can be used as researchtools for evaluating other chemical compounds. In this aspect of theinvention, a compound of formula I is used as a standard in an assay toallow comparison of the results obtained with a test compound and thecompound of formula I. For example, β₂ adrenergic receptor and/ormuscarinic receptor binding data (as determined, for example, by invitro radioligand displacement assays) for a test compound or a group oftest compounds is compared to the β₂ adrenergic receptor and/ormuscarinic receptor binding data for a compound of formula I to identifythose test compounds that have desirable binding, i.e. test compoundshaving binding about equal or superior to a compound of formula I, ifany. Alternatively, for example, bronchoprotective effects can bedetermined for test compounds and a compound of formula I in abronchoprotection assay in a mammal and this data compared to identifytest compounds providing about equal or superior bronchoprotectiveeffects. This aspect of the invention includes, as separate embodiments,both (i) the generation of comparison data (using the appropriateassays) and (ii) the analysis of the test data to identify testcompounds of interest.

The properties and utility of the compounds of this invention can bedemonstrated using various in vitro and in vivo assays well known tothose of ordinary skill in the art. For example, representative assaysare described in further detail in the following Examples.

EXAMPLES

The following Preparations and Examples are provided to illustratespecific embodiments and aspects of this invention. The illustration ofspecific embodiments and aspects, however, is not intended to limit thescope of this invention in any way unless specifically indicated.

All reagents, starting materials and solvents used in the followingexamples were purchased from commercial suppliers (such as Aldrich,Fluka, Sigma and the like) and were used without further purificationunless otherwise indicated.

In the following examples, HPLC analysis was typically conducted usingan Agilent (Palo Alto, Calif.) Series 1100 instrument with Zorbax BonusRP 2.1×50 mm columns, supplied by Agilent, (a C14 column), having a 3.5micron particle size. Detection was by UV absorbance at 214 nm. Mobilephase “A” was 2% acetonitrile, 97.9% water, and 0.1% trifluoroaceticacid (v/v/v); and mobile phase “B” was 89.9% acetonitrile, 10% water,and 0.1% trifluoroacetic acid (v/v/v). HPLC (10-70) data were obtainedwith a flow rate of 0.5 mL/minute of 10% to 70% mobile phase B gradientover a 6-minute period; HPLC (5-35) data were obtained with a flow rateof 0.5 mL/minute of 5% to 35% mobile phase B gradient over a 5-minuteperiod; and HPLC (10-90) data were obtained with a flow rate of 0.5mL/minute of 10% to 90% mobile phase B gradient over a 5-minute period.

Liquid chromatography mass spectrometry (LCMS) data typically wereobtained with an Applied Biosystems (Foster City, Calif.) ModelAPI-150EX instrument. LCMS 10-90 data were obtained with a 10% to 90%mobile phase B gradient over a 5-minute period.

Small-scale purifications were typically conducted using an API 150EXPrep Workstation system from Applied Biosystems. The mobile phase “A”was water containing 0.05% trifluoroacetic acid (v/v); and mobile phase“B” was acetonitrile containing 0.05% trifluoroacetic acid (v/v). Forsmall samples (about 3 to 50 mg recovered sample size), the followingconditions were typically used: 20 mL/min flow rate; 15 min gradientsand a 20 mm×50 mm Prism RP column with 5 micron particles (ThermoHypersil-Keystone, Bellefonte, Pa.). For larger samples (i.e., greaterthan about 100 mg crude sample), the following conditions were typicallyused: 60 mL/min flow rate; 30 min gradients and a 41.4 mm×250 mmMicrosorb BDS column with 10 micron particles (Varian, Palo Alto,Calif.).

For ¹H NMR data, the following abbreviations are used: s=singlet,d=doublet, t=triplet, q=quartet, m=multiplet, br=broad, nd=notdetermined.

Example 1 Biphenyl-2-ylcarbamic Acid Piperidin-4-yl Ester

Biphenyl-2-isocyanate (97.5 g, 521 mmol) and4-hydroxy-1-benzylpiperidine (105 g, 549 mmol) (both commerciallyavailable from Aldrich, Milwaukee, Wis.) were heated together at 70° C.for 12 h, during which time the formation of biphenyl-2-ylcarbamic acid1-benzylpiperidin-4-yl ester was monitored by LCMS. The reaction mixturewas then cooled to 50° C. and ethanol (1 L) was added, and then 6Mhydrochloric acid (191 mL) was added slowly. The reaction mixture wasthen cooled to ambient temperature and ammonium formate (98.5 g, 1.56mol) was added and nitrogen gas was bubbled through the solutionvigorously for 20 min. Palladium (10 wt. % (dry basis) on activatedcarbon) (20 g) was then added. The reaction mixture was heated at 40° C.for 12 h and then filtered through a pad of Celite. The solvent was thenremoved under reduced pressure and 1M hydrochloric acid (40 mL) wasadded to the crude residue. Sodium hydroxide (10N) was then added toadjust the pH to 12. The aqueous layer was extracted with ethyl acetate(2×150 mL) and dried (magnesium sulfate), and then the solvent wasremoved under reduced pressure to give biphenyl-2-ylcarbamic acidpiperidin-4-yl ester (155 g, 100%). HPLC (10-70) R_(t)=2.52; MS m/z:[M+H⁺] calc'd for C₁₈H₂₀N₂O₂ 297.15; found 297.3.

Example 2 3-[4-(Biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]propionic Acid

To a solution of biphenyl-2-ylcarbamic acid piperidin-4-yl ester (50 g,67.6 mmol) in dichloromethane (500 mL) was added acrylic acid (15.05 mL,100 mmol). The resulting mixture was heated at 50° C. under reflux for18 hours and then the solvent was removed. Methanol (600 mL) was addedand this mixture was heated at 75° C. for 2 hours and then cooled toroom temperature to form a thick slurry. The solid was collected byfiltration, washed with methanol (50 mL) and air dried to afford3-[4-(biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]propionic acid (61 g, 96%purity) as white powder.

Example 3N-{5-[(R)-2-Amino-1-(tert-butyldimethylsilanyloxy)ethyl]-2-hydroxyphenyl)-formamideAcetic Acid Salt StepA—N-{5-[(R)-2-Benzylamino-1-(tert-butyldimethylsilanyloxy)ethyl]-2-benzyloxyphenyl}formamide

To a 500 mL three-necked round-bottomed flask was addedN-{2-benzyloxy-5-[(R)-2-bromo-1-(tert-butyldimethylsilanyloxy)ethyl]phenyl}formamide(100 g, 215 mmol) and N-methyl-2-pyrrolidone (300 mL). Benzylamine (69.4mL, 648 mol) was added and the reaction mixture was flushed withnitrogen. The reaction mixture was then heated to 90° C. and stirred forabout 8 hours. The reaction mixture was then cooled to room temperatureand water (1.5 L) and ethyl acetate (1.5 L) were added. The layers wereseparated and the organic layer was washed with water (500 mL), a 1:1mixture of water and saturated brine (500 mL total), and then again withwater (500 mL). The organic layer was then dried over anhydrousmagnesium sulfate, filtered and concentrated under reduced pressure toprovide crudeN-{5-[(R)-2-benzylamino-1-(tert-butyldimethylsilanyloxy)ethyl]-2-benzyloxyphenyl}formamide(100 g, 90% yield, 75-80% purity) as an orange-brown thick oil.

StepB—N-{5-[(R)-2-Amino-1-(tert-butyldimethylsilanyloxy)ethyl]-2-hydroxyphenyl}-formamideAcetic Acid Salt

CrudeN-{5-[(R)-2-benzylamino-1-(tert-butyldimethylsilanyloxy)ethyl]-2-benzyloxyphenyl}formamide(100 g, 194 mmol) was dissolved in methanol (1 L) and acetic acid (25mL, 291 mmol). The resulting mixture was purged with dry nitrogen andthen palladium hydroxide on carbon (20 g, 20 wt. %, about 50% water) wasadded. Hydrogen was bubbled through the reaction mixture with stirringat room temperature for about 10 hours. The mixture was then purged withdry nitrogen and the mixture was filtered through Celite. The filtratewas concentrated on a rotary evaporator and ethyl acetate (600 mL) wasadded to the residue. This mixture was stirred for about 2 hours atwhich time a thick yellow slurry had developed. The slurry was filteredand the precipitate was air dried to provideN-{5-[(R)-2-amino-1-(tert-butyldimethylsilanyloxy)ethyl]-2-hydroxyphenyl}formamideacetic acid salt (48 g, 98% purity) as a yellow-white solid. LCMS(10-70) R_(t)=3.62; [M+H⁺] found 311.3.

Example 4 Biphenyl-2-ylcarbamic Acid1-[2-(4-{[(R)-2-(3-Formylamino-4-hydroxyphenyl)-2-hydroxyethylamino]methyl}-2,5-dimethylphenylcarbamoyl)ethyl]piperidin-4-ylEster Step A—Methyl 2,5-Dimethyl-4-nitrobenzoate

To a stirred solution of 2,5-dimethyl-4-nitrobenzoic acid (480 mg, 2.4mmol) in dry methanol (8.2 mL) at 0° C. under dry nitrogen was addedthionyl chloride (0.538 mL, 7.38 mmol). The resulting mixture as allowedto warm to room temperature and stirred for about 7 hours. Additionalthionyl chloride (0.300 mL) was added and stirring was continued at roomtemperature overnight. The solvent was removed under reduced pressureand the residue was dissolved in ethyl acetate. This solution was washedwith saturated aqueous sodium bicarbonate, dried over anhydrous sodiumsulfate and concentrated under reduced pressure to provide methyl2,5-dimethyl-4-nitrobenzoate (578 mg) as a pale yellow solid. HPLC(10-70) R_(t)=4.61; ¹H NMR (300 MHz, CDCl₃) δ 2.57 (s, 3H), 2.61 (s,3H), 3.94 (s, 3H), 7.82 (s, 1H), 7.87 (s, 1H).

Step B—Methyl 4-Amino-2,5-dimethylbenzoate

To a stirred solution of methyl 2,5-dimethyl-4-nitrobenzoate (523 mg,2.5 mmol) in a 9:1 mixture of methanol and water (25 mL total) at 0° C.was added ammonium chloride (401 mg, 7.5 mmol). Zinc (1.63 g, 25 mmol)was added portionwise and the resulting mixture was stirred at roomtemperature overnight. The reaction mixture was then filtered throughCelite and the Celite pad was washed with methanol. The filtrate wasconcentrated under reduced pressure and the resulting residue wasdissolved in ethyl acetate. This solution was washed with saturatedaqueous sodium bicarbonate, dried over anhydrous sodium sulfate andconcentrated under reduced pressure to provide methyl4-amino-2,5-dimethylbenzoate (450 mg) as a yellow oil. ¹H NMR (300 MHz,CDCl₃) δ 2.14 (s, 3H), 2.53 (s, 3H), 3.83 (s, 3H), 3.85 (br s, 2H), 6.48(s, 1H), 7.72 (s, 1H).

Step C—Methyl4-{3-[4-(Biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]propionylamino}-2,5-dimethylbenzoate

To a stirred solution of3-[4-(biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]propionic acid (670 mg,1.82 mmol) and methyl 4-amino-2,5-dimethylbenzoate (390 mg, 2.18 mmol)in dichloromethane (3.6 mL) and diisopropylethylamine (0.413 mL) wasadded O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU) (829 mg, 2.18 mmol). The resulting mixturewas stirred at room temperature overnight. The mixture was then washedwith saturated aqueous sodium bicarbonate, dried over anhydrous sodiumsulfate, filtered and concentrated under reduced pressure. The residuewas purified by silica gel chromatography eluting with dichloromethanecontaining from 3% to 5% methanol to provide methyl4-{3-[4-(biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]propionylamino}-2,5-dimethylbenzoate(568 mg, 59% yield). LCMS (10-70) R_(t)=4.55; [M+H⁺] found 530.4.

Step D—Biphenyl-2-ylcarbamic Acid1-[2-(4-Hydroxymethyl-2,5-dimethylphenylcarbamoyl)ethyl]piperidin-4-ylEster

To a stirred solution of 1M lithium aluminum hydride in THF (1.52 mL,1.52 mmol) at 0° C. was added methyl4-{3-[4-(biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]propionylamino}-2,5-dimethylbenzoate(400 mg, 0.76 mmol). The resulting mixture was stirred at 0° C. for 30minutes and then a 1:1 mixture of 1M aqueous sodium hydroxide (5 mL) andwater (5 mL) was added and stirring was continued for 2 hours.Dichloromethane was added and the organic layer was separated, driedover sodium sulfate and the solvent removed under reduced pressure. Theresidue was purified by silica gel chromatography eluting withdichloromethane containing 5% methanol to provide biphenyl-2-ylcarbamicacid1-[2-(4-hydroxymethyl-2,5-dimethylphenylcarbamoyl)-ethyl]piperidin-4-ylester. LCMS (10-70) R_(t)=3.94; [M+H⁺] found 502.5.

Step E—Biphenyl-2-ylcarbamic Acid1-[2-(4-Formyl-2,5-dimethylphenylcarbamoyl)ethyl]piperidin-4-yl Ester

To a solution of biphenyl-2-ylcarbamic acid1-[2-(4-hydroxymethyl-2,5-dimethylphenylcarbamoyl)ethyl]piperidin-4-ylester (151 mg, 0.3 mmol) in dichloromethane (3 mL) at 0° C. was addeddimethyl sulfoxide (128 μL, 1.8 mmol) and diisopropylethylamine (157 μL,0.9 mmol). After 15 minutes, sulfur trioxide pyridine complex (143 mg,0.9 mmol) was added and stirring at 0° C. was continued for 1 hour.Water was added to quench the reaction and the layers were separated.The organic layer was dried over anhydrous sodium sulfate, filtered andthe solvent removed under reduced pressure to give biphenyl-2-ylcarbamicacid 1-[2-(4-formyl-2,5-dimethylphenylcarbamoyl)ethyl]piperidin-4-ylester (150 mg, 100% yield), which was used without further purification.[M+H⁺] found 500.4.

Step F—Biphenyl-2-ylcarbamic Acid1-[2-(4-{[(R)-2-(tert-Butyldimethylsilanyloxy)-2-(3-formylamino-4-hydroxyphenyl)ethylamino]methyl}-2,5-dimethylphenylcarbamoyl)ethyl]piperidin-4-ylEster

A solution of biphenyl-2-ylcarbamic acid1-[2-(4-formyl-2,5-dimethylphenylcarbamoyl)ethyl]piperidin-4-yl ester(150 mg, 0.30 mmol) andN-{5-[(R)-2-amino-1-(tert-butyldimethylsilanyloxy)ethyl]-2-hydroxyphenyl}formamide(112 mg, 0.36 mmol) in a 1:1 mixture of dichloromethane and methanol(3.0 mL total) was stirred at room temperature for 30 minutes. Sodiumtriacetoxyborohydride (191 mg, 0.9 mmol) was added and the resultingmixture was stirred at room temperature overnight. Acetic acid was addedto quench the reaction and the mixture was concentrated under reducedpressure to give biphenyl-2-ylcarbamic acid1-[2-(4-{[(R)-2-(tert-butyldimethylsilanyloxy)-2-(3-formylamino-4-hydroxyphenyl)ethylamino]methyl}-2,5-dimethylphenylcarbamoyl)-ethyl]piperidin-4-ylester, which was used without further purification. LCMS (10-70)R_(t)=4.55; [M+H⁺] found 794.6.

Step G—Biphenyl-2-ylcarbamic Acid1-[2-(4-{[(R)-2-(3-Formylamino-4-hydroxyphenyl)-2-hydroxyethylamino]methyl}-2,5-dimethylphenylcarbamoyl)ethyl]piperidin-4-ylEster

To a suspension of biphenyl-2-ylcarbamic acid1-[2-(4-{[(R)-2-(tert-butyldimethylsilanyloxy)-2-(3-formylamino-4-hydroxyphenyl)ethylamino]methyl}-2,5-dimethylphenylcarbamoyl)-ethyl]piperidin-4-ylester (238 mg, 0.30 mmol) in dichloromethane (3.0 mL) was addedtriethylamine trihydrofluoride (147 μL, 0.90 mmol). This mixture wasstirred at room temperature overnight and then the mixture wasconcentrated under reduced pressure. The residue was purified byprep-RP-HPLC (gradient: 2 to 50% acetonitrile in water with 0.05% TFA).The appropriate fractions were collected and combined and lyophilized togive biphenyl-2-ylcarbamic acid1-[2-(4-{[(R)-2-(3-formylamino-4-hydroxyphenyl)-2-hydroxyethylamino]methyl}-2,5-dimethylphenylcarbamoyl)ethyl]piperidin-4-ylester as the ditrifluoroacetate salt (50 mg, 97% purity). LPLC (2-90)R_(t)=2.76; [M+H⁺] found 680.8.

Example 5 Biphenyl-2-ylcarbamic Acid1-[2-(4-{[(R)-2-(3-Formylamino-4-hydroxyphenyl)-2-hydroxyethylamino]methyl}-2,5-dimethylphenylcarbamoyl)ethyl]piperidin-4-ylEster Step A—Dibenzyl-(4-iodo-2,5-dimethylphenyl)amine

To a 2-liter round-bottomed flask equipped with an overhead stirrer,temperature control and an addition funnel was added4-iodo-2,5-dimethylaniline (100.0 g, 0.405 mol) (from Spectra GroupLimited, Inc., Millbury, Ohio). Ethanol (1 L) and solid potassiumcarbonate (160 g, 1.159 mol) were added and then neat benzyl bromide(140 mL, 1.179 mol) was added in one portion. The resulting mixture wasstirred at 30° C. for about 18 hours at which time HPLC shows greaterthan 98% conversion. The mixture was then cooled to room temperature andhexanes (1 L) were added. This mixture was stirred for 15 minutes andthen filtered through a paper filter to removed solids and the filtercake was washed with hexanes (200 mL). Using a rotoevaporator, thevolume of the filtrate was reduced to about 500 mL and concentratedhydrochloric acid (30 mL) was added. The remaining solvent was thenremoved using a rotoevaporator. To the resulting residue was addedhexanes (500 mL) and this mixture was stirred for about 30 minutes aswhich time a free-flowing slurry had formed. The slurry was filtered andthe filter cake was washed with hexanes (200 mL) and dried to providedibenzyl-(4-iodo-2,5-dimethylphenyl)amine hydrochloride (115 g, 62%yield, 97.5% purity) as a greenish colored solid.

The dibenzyl-(4-iodo-2,5-dimethylphenyl)amine hydrochloride wastransferred to a 3 L flask and toluene (1 L) and 1 M aqueous sodiumhydroxide (1 L) were added. The resulting mixture was stirred for 1 hourand then the layers were separated. The organic layer was washed withdilute brine (500 mL) and the solvent was removed by rotoevaporation toprovide dibenzyl-(4-iodo-2,5-dimethylphenyl)amine (80 g) as a semi-solidthick oil. (Alternatively, dichloromethane can be used in place oftoluene in this step). ¹H NMR (300 MHz, DMSO-d₆) δ 2.05 (s, 3H), 2.19(s, 3H), 3.90 (s, 4H), 6.91 (s, 1H), 7.05-7.20 (m, 10H), 7.42 (s, 1H);MS [M+H⁺] found 428.

Step B—4-Dibenzylamino-2,5-dimethylbenzaldehyde Hydrochloride

To a 1-liter 3-necked round-bottomed flask equipped with an overheadstirrer, temperature control and an addition funnel was addeddibenzyl-(4-iodo-2,5-dimethylphenyl)amine (15 g, 35 mmol). Toluene (300mL) was added and the resulting mixture was stirred for about 15minutes. The reaction flask was purged with dry nitrogen and cooled toabout −20° C. and 1.6 M n-butyllithium in hexanes (33 mL, 53 mmol) wasadded dropwise via the addition funnel. During the addition, theinternal temperature of the reaction mixture was maintained below −10°C. When the addition was complete, the resulting mixture was stirred atabout −15° C. for 15 minutes. N,N-Dimethylformamide (10 mL, 129 mmol)was then added dropwise while maintaining the internal reactiontemperature below 0° C. The resulting mixture was then stirred at −20°C. to 0° C. for about 1 hour. Aqueous 1 M hydrochloric acid (200 mL) wasthen added over a 5 minute period and the resulting mixture was stirredfor 15 minutes. The layers were then separated and the organic layer waswashed with dilute brine (100 mL). The organic layer was then dried overanhydrous sodium sulfate, filtered and the solvent removed under reducedpressure to provide 4-dibenzylamino-2,5-dimethylbenzaldehydehydrochloride (11.5 g, 90% yield, 95% purity) as thick oil thatsolidified upon standing. The product contained about 3 to 5% of thedes-iodo by-product. ¹H NMR (300 MHz, CDCl₃) δ 2.42 (s, 3H), 2.50 (s,3H), 4.25 (s, 4H), 6.82 (s, 1H), 7.10-7.30 (m, 10H), 7.62 (s, 1H), 10.15(s, 1H); MS [M+H⁺] found 330.3.

Step C—4-[1,3] Dioxolan-2-yl-2,5-dimethylphenylamine

To a 500 mL round-bottomed flask was added4-dibenzylamino-2,5-dimethylbenzaldehyde hydrochloride (11.5 g, 31.4mmol) and toluene (150 mL) and the resulting mixture was stirred untilthe salt completely dissolved. The reaction flask was then purged withdry nitrogen for 5 minutes. Ethylene glycol (5.25 mL, 94.2 mmol) andp-toluenesulfonic acid (760 mg, 6.2 mmol) were added and the resultingmixture was heated at 60° C. to 80° C. for about 20 hours. The solventwas then removed slowly (over about 40 minutes) at 40° C. on a rotaryevaporator. Toluene (100 mL) was added to the residue and the solventwas again removed slowly at 40° C. on a rotary evaporator. This processwas repeated using another aliquot of toluene (100 mL) and the mixturewas evaporated to dryness. Ethyl acetate (150 mL) and saturated aqueoussodium bicarbonate (100 mL) were added to the residue and the layerswere separated. The organic layer was washed with brine (50 mL) and thendried over anhydrous sodium sulfate, filtered and concentrated underreduced pressure to provide crudedibenzyl-(4-[1,3]dioxolan-2-yl-2,5-dimethyl-phenyl)amine (11.4 g).

The crude dibenzyl-(4-[1,3]dioxolan-2-yl-2,5-dimethyl-phenyl)amine wasdissolved in a 2:1 mixture of ethanol and water (150 mL total) and theresulting mixture was purged with dry nitrogen for 5 minutes. Palladiumon carbon (2.3 g, 10 wt. % containing about 50% water) and solid sodiumbicarbonate (1.0 g) were added and the resulting mixture washydrogenated at about 1 atm of hydrogen at 25° C. to 30° C. for about 8hours. The mixture was then filtered through Celite and the filtrate wasconcentrated on a rotary evaporator to provide crude4-[1,3]dioxolan-2-yl-2,5-dimethylphenylamine (5.6 g, 92% yield) as athick oil. ¹H NMR (300 MHz, DMSO-d₆) δ 2.05 (s, 3H), 2.22 (s, 3H),3.7-3.9 (m, 4H), 3.95 (s, 4H), 5.59 (s, 1H), 6.72 (s, 1H), 7.0-7.25 (m,11H).

Step D-N-(4-[1,3] Dioxolan-2-yl-2,5-dimethylphenyl)acrylamide

To a 500 mL round-bottom flask was added crude4-[1,3]dioxolan-2-yl-2,5-dimethylphenylamine (5.6 g, 29 mmol),dichloromethane (100 mL) and diisopropylethylamine (7.6 mL, 43.5 mmol).The resulting mixture was stirred at room temperature until theingredients dissolved and then the mixture was cooled to 0° C. Acryloylchloride (2.35 mL, 29 mmol) was then added dropwise over a 5 minuteperiod. The reaction mixture was stirred at 0° C. to 5° C. for 1 hourand then water (50 mL) was added and stirring was continued for about 30minutes at which time fine solids had formed. The mixture was filteredto collect the solids. The layers of the filtrate were then separatedand the organic layer was concentrated under reduced pressure todryness. Dichloromethane (50 mL) was added to the residue and thismixture was stirred until a free-flowing slurry developed. The slurrywas filtered (using the same funnel used to collect the fine solidsabove) and the filter cake was washed with dichloromethane (10 mL) anddried to provide N-(4-[1,3]dioxolan-2-yl-2,5-dimethylphenyl)acrylamide(3.1 g, 97% purity) as a white to off-white solid.

The filtrate from above was then evaporated to dryness and methanol (10mL) was added to the residue. This mixture was stirred for 15 minutesand then the precipitate was collected by filtration, washed withmethanol (5 mL) and dried to give a second crop ofN-(4-[1,3]dioxolan-2-yl-2,5-dimethylphenyl)acrylamide (0.8 g, 95%purity). ¹H NMR (300 MHz, CD₃OD) δ 2.10 (s, 3H), 2.23 (s, 3H), 3.85-4.10(m, 4H), 5.60-6.40 (m, 3H), 5.59 (s, 1H), 7.18 (s, 1H), 7.23 (s, 1H).

Step E—Biphenyl-2-ylcarbamic Acid1-[2-(4-Formyl-2,5-dimethylphenylcarbamoyl)ethyl]piperidin-4-yl EsterHydrochloride

To a 50 mL round-bottom flask was added biphenyl-2-ylcarbamic acidpiperidin-4-yl ester (1.2 g, 4.04 mmol) andN-(4-[1,3]dioxolan-2-yl-2,5-dimethylphenyl)acrylamide (1.0 g, 4.04mmol). Ethanol (10 mL) and dichloromethane (10 mL) were added to form aslurry. The reaction mixture was heated at 45° C. to 50° C. for about 18hours and then cooled to room temperature. Aqueous 1M hydrochloric acid(10 mL) was added and the resulting mixture was stirred vigorously forabout 3 hours. Dichloromethane (10 mL) was added and the resultingmixture was stirred for about 5 minutes. The layers were then separatedand the organic layer was dried over anhydrous sodium sulfate, filteredand concentrated on a rotary evaporator to provide crudebiphenyl-2-ylcarbamic acid1-[2-(4-formyl-2,5-dimethylphenylcarbamoyl)ethyl]piperidin-4-yl esterhydrochloride (1.9 g). ¹H NMR (300 MHz, DMSO-d₆) δ 1.2-1.4 (m, 2H),1.58-1.75 (m, 2H), 2.0-2.17 (m, 2H), 2.19 (s, 3H), 2.38 (s, 3H),2.41-2.50 (m, 4H), 2.5-2.75 (m, 2H), 4.31-4.42 (m, 1H), 7.10-7.35 (m,9H), 7.55 (s, 1H), 7.75 (s, 1H), 8.59 (s, 1H), 9.82 (s, 1H), 9.98 (s,1H); MS [M+H⁺] found 500.2.

Step F—Biphenyl-2-ylcarbamic Acid1-[2-(4-{[(R)-2-(tert-Butyldimethylsilanyloxy)-2-(3-formylamino-4-hydroxyphenyl)ethylamino]methyl}-2,5-dimethylphenyl-carbamoyl)ethyl]piperidin-4-ylEster

To a 2 L three-necked round-bottom flask was added biphenyl-2-ylcarbamicacid 1-[2-(4-formyl-2,5-dimethylphenylcarbamoyl)ethyl]piperidin-4-ylester hydrochloride (38 g, 70 mmol) andN-{5-[(R)-2-amino-1-(tert-butyldimethylsilanyloxy)ethyl]-2-hydroxyphenyl}formamideacetic acid salt (33.6 g, 91 mmol). Dichloromethane (500 mL) andmethanol (500 mL) were added and the resulting mixture was stirred atroom temperature under dry nitrogen for about 3 hours. The reactionmixture was then cooled to 0° C. to 5° C. and solid sodiumtriacetoxyborohydride (44.5 g, 381 mmol) was added in portions over a 10minute period. The reaction mixture was slowly warmed from 0° C. to roomtemperature over a period of about 2 hours and then cooled to 0° C.Saturated aqueous sodium bicarbonate (500 mL) and dichloromethane (500mL) were added. This mixture was stirred thoroughly and then the layerswere separated. The organic layer was washed with brine (500 mL), driedover anhydrous sodium sulfate, filtered and concentrated under reducedpressure to give crude biphenyl-2-ylcarbamic acid1-[2-(4-{[(R)-2-(tert-butyldimethylsilanyloxy)-2-(3-formylamino-4-hydroxyphenyl)ethylamino]methyl}-2,5-dimethylphenylcarbamoyl)-ethyl]piperidin-4-ylester (55 g, 86% purity) as a yellow solid.

The crude product (30 g) was dissolved in dichloromethane containing 2%methanol (150 mL total) and loaded onto a silica gel column (300 g) thathad been packed and equilibrated with dichloromethane containing 2%methanol and 0.5% ammonium hydroxide. The product was eluted from thecolumn using dichloromethane containing 2% methanol and 0.5% ammoniumhydroxide (1 L); dichloromethane containing 4% methanol and 0.5%ammonium hydroxide (1 L) and dichloromethane containing 5% methanol and0.5% ammonium hydroxide (about 3 L). Fractions (200 mL) were collectedand those fractions having a purity greater than 90% were combined andconcentrated under reduced pressure to provide biphenyl-2-ylcarbamicacid1-[2-(4-{[(R)-2-(tert-butyldimethylsilanyloxy)-2-(3-formylamino-4-hydroxyphenyl)ethylamino]methyl}-2,5-dimethylphenylcarbamoyl)-ethyl]piperidin-4-ylester (21.6 g, 96.5% purity) as a yellowish solid. MS [M+H⁺] found794.6.

Step G—Biphenyl-2-ylcarbamic Acid1-[2-(4-{[(R)-2-(3-Formylamino-4-hydroxyphenyl)-2-hydroxyethylamino]methyl}-2,5-dimethylphenyl-carbamoyl)ethyl]piperidin-4-ylEster Hydrofluoride Salt

To a 1 L round-bottom flask was added biphenyl-2-ylcarbamic acid1-[2-(4-{[(R)-2-(tert-butyldimethylsilanyloxy)-2-(3-formylamino-4-hydroxyphenyl)ethylamino]methyl}-2,5-dimethylphenylcarbamoyl)ethyl]piperidin-4-ylester (21.5 g, 27.1 mmol) and dichloromethane (200 mL). The resultingmixture was stirred at room temperature until the ingredients dissolvedand then triethylamine trihydrofluoride (8.85 mL, 54.2 mmol) was addedand the resulting mixture was stirred at 25° C. for about 48 hours. Thesolvent was removed on a rotary evaporator to provide a thick paste.Dichloromethane (100 mL) and ethyl acetate (200 mL) were added to thepaste and the resulting mixture was stirred for 30 minutes. Theresulting slurry was slowly filtered under dry nitrogen and the filtercake was washed with a 1:2 mixture of dichloromethane and ethyl acetate(100 mL total), dried under nitrogen for 2 hours and then dried undervacuum overnight to provide biphenyl-2-ylcarbamic acid1-[2-(4-{[(R)-2-(3-formylamino-4-hydroxyphenyl)-2-hydroxyethylamino]methyl}-2,5-dimethylphenylcarbamoyl)ethyl]piperidin-4-ylester as a hydrofluoride salt (25 g, 96.9% purity) which was a hardclay-like solid. MS [M+H⁺] found 680.8.

Step H—Biphenyl-2-ylcarbamic Acid1-[2-(4-{[(R)-2-(3-Formylamino-4-hydroxyphenyl)-2-hydroxyethylamino]methyl}-2,5-dimethylphenylcarbamoyl)-ethyl]piperidin-4-ylEster

Biphenyl-2-ylcarbamic acid1-[2-(4-{[(R)-2-(3-formylamino-4-hydroxyphenyl)-2-hydroxyethylamino]methyl}-2,5-dimethylphenylcarbamoyl)ethyl]piperidin-4-ylester hydrofluoride salt (25 g) was purified on a 6-inch reverse-phasecolumn (Microsorb solid phase) in three equal batches using a 10% to 50%mixture of acetonitrile in water containing 1% trifluoroacetic acid asthe mobile phase. Fractions with greater than 99% purity were combinedand then diluted with one volume of water. The resulting mixture wascooled to 0° C. and solid sodium bicarbonate was added until the pH ofthe mixture was about 7.5 to 8.0. Within about 5 minutes, a white slurrydeveloped. The slurry was stirred for 30 minutes and then filtered. Thefilter cake was washed with water (500 mL), air dried for about 4 hoursand then dried in vacuum overnight to provide biphenyl-2-ylcarbamic acid1-[2-(4-{[(R)-2-(3-formylamino-4-hydroxyphenyl)-2-hydroxyethylamino]methyl}-2,5-dimethylphenyl-carbamoyl)ethyl]piperidin-4-ylester (12 g, 99+% purity), as a semi-crystalline free base.

Example 6 Biphenyl-2-ylcarbamic Acid1-[2-(4-{[(R)-2-(3-Formylamino-4-hydroxyphenyl)-2-hydroxyethylamino]methyl}-2,5-dimethylphenylcarbamoyl)ethyl]piperidin-4-ylEster Step A—Biphenyl-2-ylcarbamic Acid1-[2-(4-[1,3]-Dioxolan-2-yl-2,5-dimethylphenylcarbamoyl)ethyl]piperidin-4-ylEster

To a 500 mL round-bottom flask was added biphenyl-2-ylcarbamic acidpiperidin-4-yl ester (17.0 g, 58 mmol) andN-(4-[1,3]dioxolan-2-yl-2,5-dimethylphenyl)acrylamide (13.1 g, 52.9mmol). Ethanol (150 mL) and dichloromethane (150 mL) were added to forma slurry. The reaction mixture was heated at 50° C. to 55° C. for about24 hours and then cooled to room temperature. Most of the solvent wasremoved on a rotary evaporator, resulting in a thick slurry. Ethanol(reagent grade) was added to form a total volume of about 200 mL and theresulting mixture was heated to 80° C. and then cooled slowly to roomtemperature. The resulting thick white slurry was filtered, washed withethanol (20 mL) and dried in vacuum to provide biphenyl-2-ylcarbamicacid1-[2-(4-[1,3]dioxolan-2-yl-2,5-dimethylphenylcarbamoyl)ethyl]piperidin-4-ylester (23.8 g, about 98% purity) as a white solid.

Step B—Biphenyl-2-ylcarbamic Acid1-[2-(4-Formyl-2,5-dimethylphenyl-carbamoyl)ethyl]piperidin-4-yl Ester

To a 500 mL round-bottom flask was added biphenyl-2-ylcarbamic acid1-[2-(4-[1,3]dioxolan-2-yl-2,5-dimethylphenylcarbamoyl)ethyl]piperidin-4-ylester (15 g, 27.6 mmol) and acetonitrile (150 mL) to form a slurry.Aqueous 2 M hydrochloric acid (75 mL) was added and the resultingmixture was stirred at 30° C. for 1 hour. The mixture was then cooled toroom temperature and ethyl acetate (150 mL) was added. Aqueous 2 Msodium hydroxide (75 mL) was added, the pH was checked and thenadditional 2 M sodium hydroxide was added until the pH of the solutionwas in the range of 9 to 10. The layers were separated and the organiclayer was washed with diluted brine (75 mL; 1:1 brine/water), dried overanhydrous sodium sulfate and the solvent removed on a rotary evaporatorto give biphenyl-2-ylcarbamic acid1-[2-(4-formyl-2,5-dimethylphenyl-carbamoyl)ethyl]piperidin-4-yl ester(12.5 g, about 98% purity). If desired, the purity of this intermediatecan be increased by forming a slurry with ethanol (3 volumes ofethanol), heating the slurry to 80° C., and then cooling slowly to roomtemperature and isolating by filtration.

Step C—Biphenyl-2-ylcarbamic Acid1-[2-(4-{[(R)-2-(tert-Butyldimethylsilanyloxy)-2-(3-formylamino-4-hydroxyphenyl)ethylimino]methyl}-2,5-dimethyl-phenylcarbamoyl)ethyl]piperidin-4-ylEster

To a 250 mL round-bottom flask was added biphenyl-2-ylcarbamic acid1-[2-(4-formyl-2,5-dimethylphenylcarbamoyl)ethyl]piperidin-4-yl ester(7.1 g, 14.2 mmol) andN-{5-[(R)-2-amino-1-(tert-butyldimethylsilanyloxy)ethyl]-2-hydroxyphenyl}formamideacetic acid salt (5.8 g, 15.6 mmol). Methanol (100 mL) was added to forma slurry and this mixture was stirred at 45° C. to 50° C. under nitrogenfor 1 hour. The mixture was then cooled to room temperature and toluene(50 mL) was added and the solvent was removed on a rotary evaporator attemperature ranging from 35° C. to 45° C. Toluene (50 mL) was added tothe residue and the solvent was removed to provide biphenyl-2-ylcarbamicacid1-[2-(4-{[(R)-2-(tert-butyldimethylsilanyloxy)-2-(3-formylamino-4-hydroxyphenyl)ethylimino]methyl}-2,5-dimethylphenylcarbamoyl)ethyl]piperidin-4-ylester (12 g) as a yellow-orange solid.

Step D—Biphenyl-2-ylcarbamic Acid1-[2-(4-{[(R)-2-(tert-Butyldimethylsilanyloxy)-2-(3-formylamino-4-hydroxyphenyl)ethylamino]methyl}-2,5-dimethylphenyl-carbamoyl)ethyl]piperidin-4-ylEster

To a hydrogenation flask was added biphenyl-2-ylcarbamic acid1-[2-(4-{[(R)-2-(tert-butyldimethylsilanyloxy)-2-(3-formylamino-4-hydroxyphenyl)ethylimino]methyl}-2,5-dimethylphenylcarbamoyl)ethyl]piperidin-4-ylester (4.6 g) and 2-methyltetrahydrofuran (50 mL). The resulting mixturewas stirred until the solid dissolved (about 5 min.) and then themixture was purged with nitrogen. Platinum on carbon (920 mg, 5 wt. %,support activated carbon) was added and the mixture was hydrogenated at50 psi (Parr shaker) for 6 hours. The mixture was then filtered throughCelite and the Celite was washed with 2-methyltetrahyrofuran (10 mL). Tothe filtrate was added a thiopropyl-modified silica gel (20% of weightof solution, Silicycle) and this mixture was stirred at 25° C. to 30° C.for 3 hours. The mixture was then filtered through Celite andconcentrated to remove the solvent. The residue was dissolved inmethanol (5 mL per gram of residue) and then the resulting solution wasadded slowly to a vigorously stirred 1:1 mixture of saturated aqueoussodium bicarbonate and water (40 mL per gram of residue). The resultingoff-white slurry was stirred for 20 minutes and then filtered. Thefilter cake was washed with water (20 volumes), air dried for 3 hoursand then dried in vacuum at room temperature overnight to providebiphenyl-2-ylcarbamic acid1-[2-(4-{[(R)-2-(tert-butyldimethylsilanyloxy)-2-(3-formylamino-4-hydroxyphenyl)ethylamino]methyl}-2,5-dimethylphenylcarbamoyl)ethyl]piperidin-4-ylester (80% recovery, about 96% purity).

Step E—Biphenyl-2-ylcarbamic Acid1-[2-(4-{[(R)-2-(3-Formylamino-4-hydroxyphenyl)-2-hydroxyethylamino]methyl}-2,5-dimethylphenyl-carbamoyl)ethyl]piperidin-4-ylEster L-Tartrate Salt

To a 200 mL round-bottom flask was added biphenyl-2-ylcarbamic acid1-[2-(4-{[(R)-2-(tert-butyldimethylsilanyloxy)-2-(3-formylamino-4-hydroxyphenyl)ethylamino]-methyl}-2,5-dimethylphenylcarbamoyl)ethyl]piperidin-4-ylester (3.8 g, 4.8 mmol) and 2-methyltetrahydrofuran (40 mL). Theresulting mixture was stirred at room temperature until the ingredientsdissolved (about 15 min.) and then triethylamine trihydrofluoride (0.94mL, 5.76 mmol) was added and the resulting mixture was stirred at 25° C.for about 24 hours. To this mixture was added a 1:1 mixture of saturatedaqueous sodium bicarbonate and water (40 mL) and 2-methyltetrahydrofuranand the resulting mixture was stirred until the solid dissolved(solution pH about 8). The layers were separated and the organic layerwas washed with brine (30 mL), dried over anhydrous sodium sulfate,filtered and concentrated on a rotary evaporator. The residue wasdissolved in 2-methyltetrahydrofuran (50 mL) and solid L-tartaric acid(650 mg) was added. The resulting mixture was stirred at 25° C. to 30°C. for 18 hours and then filtered through filter paper. The filter cakewas washed with 2-methyltetrahydrofuran (10 mL), isopropanol (10 mL) andimmediately put under vacuum to provide biphenyl-2-ylcarbamic acid1-[2-(4-{[(R)-2-(3-formylamino-4-hydroxyphenyl)-2-hydroxyethylamino]methyl}-2,5-dimethylphenyl-carbamoyl)ethyl]piperidin-4-ylester L-tartaric acid salt (3.7 g, >97% purity).

Step F—Biphenyl-2-ylcarbamic Acid1-[2-(4-{[(R)-2-(3-Formylamino-4-hydroxyphenyl)-2-hydroxyethylamino]methyl}-2,5-dimethylphenylcarbamoyl)-ethyl]piperidin-4-ylEster

To a 250 mL round-bottom flask was added biphenyl-2-ylcarbamic acid1-[2-(4-{[(R)-2-(3-formylamino-4-hydroxyphenyl)-2-hydroxyethylamino]methyl}-2,5-dimethylphenylcarbamoyl)ethyl]piperidin-4-ylester L-tartaric acid salt (3.5 g) and methanol (35 mL) and theresulting mixture was stirred for 15 min. A 1:1 mixture of saturatedaqueous sodium bicarbonate and water (70 mL) was added over a 5 min.period and stirring was continued for 2 hours. The resulting off-whiteslurry was filtered and the filter cake was washed with water (20 mL),air dried for 2 hours and then dried in a vacuum overnight to providebiphenyl-2-ylcarbamic acid1-[2-(4-{[(R)-2-(3-formylamino-4-hydroxyphenyl)-2-hydroxyethylamino]methyl}-2,5-dimethylphenylcarbamoyl)-ethyl]piperidin-4-ylester (2.3 g) as a semi-crystalline free base.

¹H and ¹³C NMR spectra were obtained for a sample ofbiphenyl-2-ylcarbamic acid1-[2-(4-{[(R)-2-(3-formylamino-4-hydroxyphenyl)-2-hydroxyethylamino]methyl}-2,5-dimethylphenylcarbamoyl)-ethyl]piperidin-4-ylester (22.2 mg in about 0.75 mL of DMSO-d₆) at ambient temperature usinga JEOL ECX-400 NMR spectrometer:

¹H NMR (400 MHz, DMSO-d₆), major isomer, 69.64 (br, 1H), 9.54 (br s,1H), 9.43 (s, 1H), 8.67 (s, 1H), 8.26 (s, 1H), 8.03 (d, J=1.9, 1H),7.25-7.45 (m, 9H), ˜7.3 (nd, 1H), 7.07 (s, 1H), 6.88 (dd, J=8.2, 1.9,1H), 6.79 (d, J=8.2, 1H), 5.15 (br, 1H), 4.53 (dd, J=7.3, 4.7, 1H), 4.47(m, 1H), ˜3.65 and 3.60 (AB pair, 2H), 2.68 (br m, 2H), ˜2.59 (nd, 4H),2.44 (br t, J=6.5, 2H), 2.20 (s, 3H), ˜2.17 (br m, 2H), 2.14 (s, 3H),1.73 (br, 2H), 1.44 (br q, J=9.0, 2H).

¹H NMR (400 MHz, DMSO-d₆), minor isomer, 69.64 (br, 1H), 9.43 (s 1H),9.26 (br d, J=7.0, 1H), 8.67 (s, 1H), 8.50 (br d, J=7.0, 1H), 7.25-7.45(m, 9H), 7.3 (nd, 1H), 7.07 (s, 1H), ˜7.07 (nd, 1H), 6.95 (dd, J=8.3,1.8, 1H), 6.83 (d, J=8.3, 1H), 5.15 (br, 1H), 4.47 (m, 1H), 3.65 and˜3.60 (AB pair, 2H), 2.68 (br m, 2H), ˜2.59 (nd, 2H), 2.44 (br t, J=6.5,2H), 2.20 (s, 3H), ˜2.17 (br m, 2H), 2.14 (s, 3H), 1.73 (br, 2H), 1.44(br q, J=˜9.0, 2H).

¹³C NMR (100 MHz, DMSO-d₆), major isomer, δ 170.0, 159.9, 153.9, 145.5,139.3, 137.6, 135.2, 135.0, 134.8, 133.4, 133.4, 130.2, 130.2, 128.6,128.2, 127.8, 127.4, 127.2, 127.0, 126.1, 125.7, 125.6, 121.7, 118.6,114.5, 71.4, 70.0, 57.4, 53.9, 50.3, 50.1, 33.7, 30.7, 18.2, 17.5.

¹³C NMR (100 MHz, DMSO-d₆), minor isomer, δ 170.0, 163.4, 153.9, 147.8,139.3, 137,6, 135.7, 135.2, 135.0, 134.8, 133.4, 133.4, 130.2, 130.2,128.6, 128.2, 127.8, 127.4, 127.2, 127.0, 126.1, 125.7, 123.0, 119.6,115.6, 71.0, 70.0, 57.3, 53.9, 50.3, 50.1, 33.7, 30.7, 18.2, 17.5.

The ¹H and ¹³C NMR spectra showed the presence of a major isomer (about82 mole percent) and a minor isomer (about 18 mole percent) believed tobe rotational isomers resulting from hindered rotation about the—NH—C(O)H bond. The phenyl group is believed to be syn to the carbonyloxygen in the major isomer and anti in the minor isomer.

Example 7 Seed Crystals of Form II of Biphenyl-2-ylcarbamic Acid1-[2-(4-{[(R)-2-(3-Formylamino-4-hydroxyphenyl)-2-hydroxyethylamino]methyl}-2,5-dimethylphenylcarbamoyl)ethyl]piperidin-4-ylEster

Semi-crystalline biphenyl-2-ylcarbamic acid1-[2-(4-{[(R)-2-(3-formylamino-4-hydroxyphenyl)-2-hydroxyethylamino]methyl}-2,5-dimethylphenyl-carbamoyl)ethyl]piperidin-4-ylester (500 mg) was dissolved in methanol (50 mL) and water was addeduntil the cloud point was reached. The resulting mixture was stirred at25° C. for 3 hours and the resulting crystalline material was isolatedby filtration to provide crystalline biphenyl-2-ylcarbamic acid1-[2-(4-{[(R)-2-(3-formylamino-4-hydroxyphenyl)-2-hydroxyethylamino]methyl}-2,5-dimethylphenylcarbamoyl)ethyl]piperidin-4-ylester (420 mg). This crystalline free base was determined to have adifferential scanning calorimetry (DSC) trace that exhibit a peak inendothermic heat flow at about 142° C. to about 150° C.; and a powderx-ray diffraction (PXRD) pattern having significant diffraction peaks,among other peaks, at 20 values of about 20.7±0.3, 21.6±0.3, 22.5±0.3and 23.2±0.3. This crystalline free base form is designated as Form II.Additional information on Form II and other crystalline free base formsof this compound is disclosed in commonly-assigned U.S. Application No.______, filed on even date herewith (Attorney Docket No. P-222-US1) andU.S. Provisional Application No. 60/794,709, filed Apr. 25, 2006; thedisclosures of which are incorporated herein by reference in theirentirety.

Example 8 Crystallization of Form II of Biphenyl-2-ylcarbamic acid1-[2-(4-{[(R)-2-(3-Formylamino-4-hydroxyphenyl)-2-hydroxyethylamino]methyl}-2,5-dimethylphenylcarbamoyl)ethyl]piperidin-4-ylEster

To a 3 L three-necked round-bottomed flask equipped with an over-headstirrer, temperature control and addition funnel was addedsemi-crystalline biphenyl-2-ylcarbamic acid1-[2-(4-{[(R)-2-(3-formylamino-4-hydroxyphenyl)-2-hydroxyethylamino]methyl}-2,5-dimethylphenylcarbamoyl)ethyl]piperidin-4-ylester (14 g) and methanol (1.4 L). Water (500 mL) was added in oneportion and then additional water (200 mL) was added slowly until thecloud point was reached. Seed crystals of biphenyl-2-ylcarbamic acid1-[2-(4-{[(R)-2-(3-formylamino-4-hydroxyphenyl)-2-hydroxyethylamino]methyl}-2,5-dimethylphenyl-carbamoyl)ethyl]piperidin-4-ylester Form II (50 mg) were added and the resulting mixture was stirredat 25° C. for 3 hours at which time a free-flowing slurry had developed.Water (300 mL) was added over a 15-minute period and the resultingmixture was stirred at 25° C. overnight. The mixture was then filteredand the filter cake was washed with water (100 mL), air dried for about2 hours and then dried under vacuum at room temperature for 48 hours toprovide crystalline biphenyl-2-ylcarbamic acid1-[2-(4-{[(R)-2-(3-formylamino-4-hydroxyphenyl)-2-hydroxyethylamino]methyl}-2,5-dimethylphenylcarbamoyl)ethyl]piperidin-4-ylester (12.5 g, 99.6% purity). This crystalline salt was determined to beForm II.

Example 9 Biphenyl-2-ylcarbamic Acid1-[2-(4-Formyl-2,5-dimethylphenylcarbamoyl)ethyl]-piperidin-4-yl EsterStep A—4-Iodo-2,5-dimethylphenylamine

To a solution of 2,5-dimethylaniline (20 g, 165 mmol) in a 1:1 mixtureof dichloromethane and methanol (400 mL) was added sodium bicarbonate(20.8 g, 250 mmol) and tetramethylammonium dichloroiodate(I) (44.7 g,165 mmol). The resulting mixture was stirred at room temperature for 1hour and then water was added (500 mL). The organic layer was removedand washed with 5% aqueous sodium thiosulfate (500 mL) and brine (500mL). The organic layer was then dried over anhydrous magnesium sulfate,filtered and concentrated under vacuum to provide4-iodo-2,5-dimethylphenylamine (39.6 g, 98% yield). The product was usedwithout further purification.

Step B—N-(4-Iodo-2,5-dimethylphenyl)acrylamide

To a solution of 4-iodo-2,5-dimethylphenylamine (37.2 g, 151 mmol) indichloromethane (500 mL) was added sodium bicarbonate (25.4 g, 302mmol). The resulting mixture was cooled to 0° C. and acryolyl chloride(12.3 mL, 151 mmol) was added slowly over a period of 25 minutes. Theresulting mixture was stirred at room temperature overnight and thenfiltered. The volume of the filtrate was reduced to about 100 mL and aprecipitate formed. The precipitate was filtered, dried, washed withwater (1 L) and then dried again to affordN-(4-iodo-2,5-dimethylphenyl)acrylamide (42.98 g, 95% purity, 90%yield). The product was used without further purification.

Step C—Biphenyl-2-ylcarbamic Acid1-[2-(4-Iodo-2,5-dimethylphenylcarbamoyl)-ethyl]piperidin-4-yl Ester

To a solution of N-(4-iodo-2,5-dimethylphenyl)acrylamide (32.2 g, 107mmol) in a 6:1 v/v mixture of N,N-dimethylformamide and isopropanol (700mL) was added biphenyl-2-ylcarbamic acid piperidin-4-yl ester (36.3 g,123 mmol). The resulting mixture was heated at 50° C. for 24 hours andthen at 80° C. for 24 hours. The reaction mixture was then cooled toroom temperature and concentrated under vacuum. The residue wasdissolved in dichloromethane (1 L) and this solution was washed with 1Naqueous hydrochloric acid (500 mL), water (500 mL), brine (500 mL) andsaturated aqueous sodium bicarbonate solution (500 mL). The organiclayer was then dried over anhydrous magnesium sulfate and filtered.Ethanol (400 mL) was added and the resulting mixture was concentratedunder vacuum to a volume of about 400 mL, at which time a precipitatehad formed. The precipitate was filtered and dried to affordbiphenyl-2-ylcarbamic acid1-[2-(4-iodo-2,5-dimethylphenylcarbamoyl)ethyl]piperidin-4-yl ester(59.6 g, 84% purity, 79% yield). m/z: [M+H⁺] calcd for C₂₉H₃₂IN₃O₃598.49; found 598.5.

StepD—4-{3-[4-(Biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]propionylamino}-2,5-dimethylbenzoicAcid Methyl Ester

To a solution of biphenyl-2-ylcarbamic acid1-[2-(4-iodo-2,5-dimethylphenylcarbamoyl)ethyl]piperidin-4-yl ester (56g, 94 mmol) in a 5:1 v/v mixture of N,N-dimethylformamide and methanol(600 mL) were added diispropylethylamine (49 mL, 281 mmol),1,3-bis(diphenylphosphino)propane (3.9 g, 9.4 mmol) and palladium(II)acetate (2.1 g, 9.4 mmol). The resulting mixture was purged with carbonmonoxide and then stirred overnight at 70° C. to 80° C. under a carbonmonoxide atmosphere (balloon pressure). The reaction mixture wasconcentrated under vacuum and the residue was dissolved indichloromethane (500 mL). This mixture was washed with 1N aqueoushydrochloric acid (500 mL), water (500 mL) and then brine (500 mL). Theorganic layer was then dried over anhydrous magnesium sulfate, filteredand then concentrated under vacuum. The residue was mixed with ethanol(about 5:1 v/w ethanol to residue) and the mixture was heated until allsolid material dissolved. This solution was allowed to slowly cool toroom temperature and the resulting precipitate was isolated byfiltration to afford4-{3-[4-(biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]propionylamino}-2,5-dimethylbenzoicacid methyl ester (47.3 g, 97% purity, 92% yield). m/z: [M+H⁺] calcd forC₃₁H₃₅N₃O₅ 530.63; found 530.4.

Step E—Biphenyl-2-ylcarbamic Acid1-[2-(4-Hydroxymethyl-2,5-dimethyl-phenylcarbamoyl)ethyl]piperidin-4-ylEster

To a solution of4-{3-[4-(biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]propionylamino}-2,5-dimethylbenzoicacid methyl ester (49.8 g, 93.9 mmol) in tetrahydrofuran (200 mL) wascooled to 0° C. and lithium aluminum hydride (10.7 g, 281.7 mmol) addedportion-wise (10×1.07 g). The resulting mixture was stirred for 3 hoursand then water (10.7 mL) was added, followed by 1N aqueous sodiumhydroxide (10.7 mL) and additional water (32.1 mL). This mixture wasstirred overnight and then filtered. The organic layer was concentratedunder vacuum and the residue was mixed with ethyl acetate (about 5:1 v/wethyl acetate to residue). This mixture was heated until all the solidmaterial dissolved and then the solution was allowed to cool to roomtemperature. The resulting precipitate was filtered and dried to affordbiphenyl-2-ylcarbamic acid1-[2-(4-hydroxymethyl-2,5-dimethylphenylcarbamoyl)ethyl]piperidin-4-ylester (24.6 g, 95% purity, 47.5% yield). This material was used withoutfurther purification. m/z: [M+H⁺] calcd for C₃₀H₃₅N₃O₄ 502.62; found502.5.

Step F—Biphenyl-2-ylcarbamic Acid1-[2-(4-Formyl-2,5-dimethylphenylcarbamoyl)ethyl]piperidin-4-yl Ester

To a solution of biphenyl-2-ylcarbamic acid1-[2-(4-hydroxymethyl-2,5-dimethylphenylcarbamoyl)ethyl]piperidin-4-ylester (5.0 g, 10 mmol) in dichloromethane (200 mL) was addeddiisopropylethylamine (8.7 mL, 50 mmol) and dimethylsulfoxide (5.6 mL,100 mmol). The resulting mixture was cooled to 0° C. and sulfur trioxidepyridine complex (8.0 g, 50 mmol) was added. The reaction mixture wasstirred for 1 hour at 0° C. and then water (300 mL) was added. Theorganic layer was removed and washed with 1N aqueous hydrochloric acid(300 mL) and brine (300 mL). The organic layer was then dried overanhydrous magnesium sulfate and filtered. The resulting solutioncontaining biphenyl-2-ylcarbamic acid1-[2-(4-formyl-2,5-dimethylphenylcarbamoyl)ethyl]piperidin-4-yl esterwas used without further purification. m/z: [M+H⁺ ] calcd for C₃₀H₃₃N₃O₄500.60; found 500.4.

Example 10 Cell Culture and Membrane Preparation From Cells ExpressingHuman M₁, M₂, M₃ and M₄ Muscarinic Receptors

CHO cell lines stably expressing cloned human hM₁, hM₂, hM₃ and hM₄muscarinic receptor subtypes, respectively, were grown to nearconfluency in Hams F-12 media supplemented with 10% FBS and 250 μg/mLGeneticin. The cells were grown in a 5% CO₂, 37° C. incubator and liftedwith 2 mM EDTA in dPBS. Cells were collected by 5 minute centrifugationat 650×g, and cell pellets were either stored frozen at −80 EC ormembranes were prepared immediately for use. For membrane preparation,cell pellets were resuspended in lysis buffer and homogenized with aPolytron PT-2100 tissue disrupter (Kinematica AG; 20 seconds×2 bursts).Crude membranes were centrifuged at 40,000×g for 15 minutes at 4° C. Themembrane pellet was then resuspended with re-suspension buffer andhomogenized again with the Polytron tissue disrupter. The proteinconcentration of the membrane suspension was determined by the methoddescribed in Lowry et al., 1951, Journal of Biochemistry, 193, 265. Allmembranes were stored frozen in aliquots at −80° C. or used immediately.Aliquots of prepared hM₅ receptor membranes were purchased directly fromPerkinElmer, Inc. (Wellesley, Mass.) and stored at −80° C. until use.

Example 11 Radioligand Binding Assay for Muscarinic Receptors

Radioligand binding assays for cloned muscarinic receptors wereperformed in 96-well microtiter plates in a total assay volume of 100μL. CHO cell membranes stably expressing either the hM₁, hM₂, hM₃, hM₄or hM₅ muscarinic subtype were diluted in assay buffer to the followingspecific target protein concentrations (μg/well): 10 μg for hM₁, 10-15μg for hM₂, 10-20 μg for hM₃, 10-20 μg for hM₄, and 10-12 μg for hM₅ toget similar signals (cpm). The membranes were briefly homogenized usinga Polytron tissue disruptor (10 seconds) prior to assay plate addition.Saturation binding studies for determining K_(D) values of theradioligand were performed using L-[N-methyl-³H]scopolamine methylchloride ([³H]-NMS) (TRK666, 84.0 Ci/mmol, Amersham Pharmacia Biotech,Buckinghamshire, England) at concentrations ranging from 0.001 nM to 20nM. Displacement assays for determination of K_(i) values of testcompounds were performed with [³H]-NMS at 1 nM and eleven different testcompound concentrations. The test compounds were initially dissolved toa concentration of 400 μM in dilution buffer and then serially diluted5× with dilution buffer to final concentrations ranging from 10 pM to100 μM. The addition order and volumes to the assay plates were asfollows: 25 μL radioligand, 25 μL diluted test compound, and 50 μLmembranes. Assay plates were incubated for 60 minutes at 37° C. Bindingreactions were terminated by rapid filtration over GF/B glass fiberfilter plates (PerkinElmer, Inc.) pre-treated in 1% BSA. Filter plateswere rinsed three times with wash buffer (10 mM HEPES) to remove unboundradioactivity. The plates were then air-dried and 50 μL Microscint-20liquid scintillation fluid (PerkinElmer, Inc.) were added to each well.The plates were then counted in a PerkinElmer Topcount liquidscintillation counter (PerkinElmer, Inc.). Binding data were analyzed bynonlinear regression analysis with the GraphPad Prism Software package(GraphPad Software, Inc., San Diego, Calif.) using the one-sitecompetition model. K_(i) values for test compounds were calculated fromobserved IC₅₀ values and the K_(D) value of the radioligand using theCheng-Prusoff equation (Cheng Y; Prusoff W H. (1973) BiochemicalPharmacology, 22(23):3099-108). K_(i) values were converted to pK_(i)values to determine the geometric mean and 95% confidence intervals.These summary statistics were then converted back to K_(i) values fordata reporting.

In this assay, a lower K_(i) value indicates that the test compound hasa higher binding affinity for the receptor tested. Biphenyl-2-ylcarbamicacid1-[2-(4-{[(R)-2-(3-formylamino-4-hydroxyphenyl)-2-hydroxyethylamino]methyl}-2,5-dimethylphenylcarbamoyl)ethyl]piperidin-4-ylester (compound IIa) was found to have a K_(i) value of less than 10 nMfor the M₁, M₂, M₃, M₄ and M₅ muscarinic receptor subtypes.

Example 12 Cell Culture and Membrane Preparation From Cells ExpressingHuman β₁, β₂ or β₃ Adrenergic Receptors

Human embryonic kidney (HEK-293) cell lines stably expressing clonedhuman β₁ and β₂ adrenergic receptors or Chinese hamster ovarian (CHO)cell lines stably expressing cloned human β₃ adrenergic receptors weregrown to near confluency in DMEM or Hams F-12 media with 10% FBS in thepresence of 500 μg/mL Geneticin. The cell monolayer was lifted with 2 mMEDTA in PBS. Cells were pelleted by centrifugation at 1,000 rpm, andcell pellets were either stored frozen at −80° C. or membranes wereprepared immediately for use. For preparation of β₁ and β₂ receptorexpressing membranes, cell pellets were re-suspended in lysis buffer (10mM HEPES/HCl, 10 mM EDTA, pH 7.4 at 4° C.) and homogenized using atight-fitting Dounce glass homogenizer (30 strokes) on ice. For the moreprotease-sensitive β₃ receptor expressing membranes, cell pellets werehomogenated in lysis buffer (10 mM Tris/HCl, pH 7.4) supplemented withone tablet of “Complete Protease Inhibitor Cocktail Tablets with 2 mMEDTA” per 50 mL buffer (Roche Molecular Biochemicals, Indianapolis,Ind.). The homogenate was centrifuged at 20,000×g, and the resultingpellet was washed once with lysis buffer by re-suspension andcentrifugation as above. The final pellet was then re-suspended inice-cold binding assay buffer (75 mM Tris/HCl pH 7.4, 12.5 mM MgCl₂, 1mM EDTA). The protein concentration of the membrane suspension wasdetermined by the methods described in Lowry et al., 1951, Journal ofBiological Chemistry, 193, 265; and Bradford, Analytical Biochemistry,1976, 72, 248-54. All membranes were stored frozen in aliquots at −80°C. or used immediately.

Example 13 Radioligand Binding Assay for Human β₁, β₂ and β₃ AdrenergicReceptors

Binding assays were performed in 96-well microtiter plates in a totalassay volume of 100 μL with 10-15 μg of membrane protein containing thehuman β₁, β₂ or β₃ adrenergic receptors in assay buffer (75 mM Tris/HClpH 7.4 at 25° C., 12.5 mM MgCl₂, 1 mM EDTA, 0.2% BSA). Saturationbinding studies for determining K_(d) values of the radioligand weredone using [³H]-dihydroalprenolol (NET-720, 100 Ci/mmol, PerkinElmerLife Sciences Inc., Boston, Mass.) for the β₁ and β₂ receptors and[¹²⁵I]-(−)-iodocyanopindolol (NEX-189, 220 Ci/mmol, PerkinElmer LifeSciences Inc., Boston, Mass.) at 10 or 11 different concentrationsranging from 0.01 nM to 20 nM. Displacement assays for determination ofK_(i) values of test compounds were done with [³H]-dihydroalprenolol at1 nM and [¹²⁵I]-(−)-iodocyanopindolol at 0.5 nM for 10 or 11 differentconcentrations of test compound ranging from 10 pM to 10 μM.Non-specific binding was determined in the presence of 10 μMpropranolol. Assays were incubated for 1 hour at 37° C., and thenbinding reactions were terminated by rapid filtration over GF/B for theβ₁ and β₂ receptors or GF/C glass fiber filter plates for the β₃receptors (Packard BioScience Co., Meriden, Conn.) presoaked in 0.3%polyethyleneimine. Filter plates were washed three times with filtrationbuffer (75 mM Tris/HCl pH 7.4 at 4° C., 12.5 mM MgCl₂, 1 mM EDTA) toremove unbound radioactivity. The plates were then dried and 50 μL ofMicroscint-20 liquid scintillation fluid (Packard BioScience Co.,Meriden, Conn.) was added and plates were counted in a Packard Topcountliquid scintillation counter (Packard BioScience Co., Meriden, Conn.).Binding data were analyzed by nonlinear regression analysis with theGraphPad Prism Software package (GraphPad Software, Inc., San Diego,Calif.) using the 3-parameter model for one-site competition. The curveminimum was fixed to the value for nonspecific binding, as determined inthe presence of 10 μM propranolol. K_(i) values for test compounds werecalculated from observed IC₅₀ values and the K_(d) value of theradioligand using the Cheng-Prusoff equation (Cheng Y, and Prusoff W H.,Biochemical Pharmacology, 1973, 22, 23, 3099-108).

In this assay, a lower K_(i) value indicates that a test compound has ahigher binding affinity for the receptor tested. Biphenyl-2-ylcarbamicacid1-[2-(4-{[(R)-2-(3-formylamino-4-hydroxyphenyl)-2-hydroxyethylamino]methyl}-2,5-dimethylphenylcarbamoyl)ethyl]piperidin-4-ylester (compound IIa) was found to have a K_(i) value of less than 10 nMfor the β₂ adrenergic receptor and K_(i) values greater than 1000 nM forthe β₁ and β₃ adrenergic receptors.

Example 14 Functional Assays of Antagonism for Muscarinic ReceptorSubtypes

Assay A—Blockade of Agonist-Mediated Inhibition of cAMP Accumulation

In this assay, the functional potency of a test compound as anantagonist for the hM₂ receptor was determined by measuring the abilityof the test compound to block oxotremorine-inhibition offorskolin-mediated cAMP accumulation in CHO-K1 cells expressing the hM₂receptor. cAMP assays were performed in a radioimmunoassay format usingthe Flashplate Adenylyl Cyclase Activation Assay System with ¹²⁵I-cAMP(NEN SMP004B, PerkinElmer Life Sciences Inc., Boston, Mass.), accordingto the manufacturer's instructions. Cells were rinsed once with dPBS andlifted with Trypsin-EDTA solution (0.05% trypsin/0.53 mM EDTA) asdescribed in the Cell Culture and Membrane Preparation section above.The detached cells were washed twice by centrifugation at 650×g for fiveminutes in 50 mL dPBS. The cell pellet was then re-suspended in 10 mLdPBS, and the cells were counted with a Coulter Z1 Dual Particle Counter(Beckman Coulter, Fullerton, Calif.). The cells were centrifuged againat 650×g for five minutes and re-suspended in stimulation buffer to anassay concentration of 1.6×10⁶ to 2.8×10⁶ cells/mL.

The test compound was initially dissolved to a concentration of 400 μMin dilution buffer (dPBS supplemented with 1 mg/mL BSA (0.1%)), and thenserially diluted with dilution buffer to final molar concentrationsranging from 100 μM to 0.11 nM. Oxotremorine was diluted in a similarmanner.

To measure oxotremorine inhibition of adenylyl cyclase activity, 25 μLforskolin (25 μM final concentration diluted in dPBS), 25 μL dilutedoxotremorine, and 50 μL cells were added to agonist assay wells. Tomeasure the ability of a test compound to block oxotremorine-inhibitedadenylyl cyclase activity, 25 μL forskolin and oxotremorine (25 μM and 5μM final concentrations, respectively, diluted in dPBS), 25 μL dilutedtest compound, and 50 μL cells were added to remaining assay wells.

Reactions were incubated for 10 minutes at 37° C. and stopped byaddition of 100 μL ice-cold detection buffer. Plates were sealed,incubated overnight at room temperature and counted the next morning ona PerkinElmer TopCount liquid scintillation counter (PerkinElmer Inc.,Wellesley, Mass.). The amount of cAMP produced (pmol/well) wascalculated based on the counts observed for the samples and cAMPstandards, as described in the manufacturer's user manual. Data wasanalyzed by nonlinear regression analysis with the GraphPad PrismSoftware package (GraphPad Software, Inc., San Diego, Calif.) using thenon-linear regression, one-site competition equation. The Cheng-Prusoffequation was used to calculate the K_(obs), using the EC₅₀ of theoxotremorine concentration-response curve and the oxotremorine assayconcentration as the K_(D) and [L], respectively.

In this assay, a lower K_(obs) value indicates that the test compoundhas a higher functional activity at the receptor tested.Biphenyl-2-ylcarbamic acid1-[2-(4-{[(R)-2-(3-formylamino-4-hydroxyphenyl)-2-hydroxyethylamino]methyl}-2,5-dimethylphenylcarbamoyl)ethyl]piperidin-4-ylester (compound IIa) was found to have a K_(obs) value of less thanabout 10 nM for blockade of oxotremorine-inhibition offorskolin-mediated cAMP accumulation in CHO-K1 cells expressing the hM₂receptor.

Assay B—Blockade of Agonist-Mediated [³⁵S]GTPγS Binding

In this functional assay, the functional potency of a test compound asan antagonist of the hM₂ receptor was determined by measuring theability of the test compound to block oxotremorine-stimulated [³⁵S]GTPγSbinding in CHO-K1 cells expressing the hM₂ receptor.

At the time of use, frozen membranes were thawed and then diluted inassay buffer with a final target tissue concentration of 5 to 10 μgprotein per well. The membranes were briefly homogenized using aPolytron PT-2100 tissue disrupter and then added to the assay plates.

The EC₉₀ value (effective concentration for 90% maximal response) forstimulation of [³⁵S]GTPγS binding by the agonist oxotremorine wasdetermined in each experiment.

To determine the ability of a test compound to inhibitoxotremorine-stimulated [³⁵S]GTPγS binding, the following was added toeach well of 96 well plates: 25 μL of assay buffer with [³⁵S]GTPγS (0.4nM), 25 μL of oxotremorine(EC₉₀) and GDP (3 uM), 25 μL of diluted testcompound and 25 μL CHO cell membranes expressing the hM₂ receptor. Theassay plates were then incubated at 37° C. for 60 minutes. The assayplates were filtered over 1% BSA-pretreated GF/B filters using aPerkinElmer 96-well harvester. The plates were rinsed with ice-cold washbuffer for 3×3 seconds and then air or vacuum dried. Microscint-20scintillation liquid (50 μL) was added to each well, and each plate wassealed and radioactivity counted on a Topcounter (PerkinElmer). Datawere analyzed by nonlinear regression analysis with the GraphPad PrismSoftware package (GraphPad Software, Inc., San Diego, Calif.) using thenon-linear regression, one-site competition equation. The Cheng-Prusoffequation was used to calculate the K_(obs), using the IC₅₀ values of theconcentration-response curve for the test compound and the oxotremorineconcentration in the assay as the K_(D) and [L], ligand concentration,respectively.

In this assay, a lower K_(obs) value indicates that the test compoundhas a higher functional activity at the receptor tested.Biphenyl-2-ylcarbamic acid1-[2-(4-{[(R)-2-(3-formylamino-4-hydroxyphenyl)-2-hydroxyethylamino]methyl}-2,5-dimethylphenylcarbamoyl)ethyl]piperidin-4-ylester (compound IIa) was found to have a K_(obs) value of less thanabout 10 nM for blockade of oxotremorine-stimulated [³⁵S]GTPγS bindingin CHO-K1 cells expressing the hM₂ receptor.

Assay C—Blockade of Agonist-Mediated Calcium Release via FLIPR Assays

In this functional assay, the functional potency of a test compound asan antagonist of hM₁, hM₃ and cM₅ receptors was determined by measuringthe ability of the test compound to inhibit agonist-mediated increasesin intracellular calcium.

CHO cells stably expressing the receptors were seeded into 96-well FLIPRplates the night before the assay was done. Seeded cells were washedtwice with FLIPR buffer (10 mM HEPES, pH 7.4, 2 mM calcium chloride, 2.5mM probenecid in Hank's Buffered Salt Solution (HBSS) without calciumand magnesium) using Cellwash (MTX Labsystems, Inc.) to remove growthmedia. After washing, each well contained 50 μL of FLIPR buffer. Thecells were then incubated with 50 μL/well of 4 μM FLUO-4 μM (a 2×solution was made) for 40 minutes at 37° C., 5% carbon dioxide.Following the dye incubation period, cells were washed two times withFLIPR buffer, leaving a final volume of 50 μL in each well.

The dose-dependent stimulation of intracellular Ca²⁺ release foroxotremorine was determined so that the test compound could be measuredagainst oxotremorine stimulation at an EC₉₀ concentration. Cells werefirst incubated with compound dilution buffer for 20 minutes and thenoxotremorine was added. An EC₉₀ value for oxotremorine was generatedaccording to the method detailed in the FLIPR measurement and datareduction section below, in conjunction with the formulaEC_(F)=((F/100-F)̂1/H)*EC₅₀. An oxotremorine concentration of 3×EC_(F)was prepared in stimulation plates such that an EC₉₀ concentration ofoxotremorine was added to each well in test assay plates.

The parameters used for the FLIPR were: exposure length of 0.4 seconds,laser strength of 0.5 watts, excitation wavelength of 488 nm, andemission wavelength of 550 nm. Baseline was determined by measuring thechange in fluorescence for 10 seconds prior to addition of oxotremorine.Following oxotremorine stimulation, the FLIPR continuously measured thechange of fluorescence every 0.5 to 1 second for 1.5 minutes to capturethe maximum fluorescence change.

The change of fluorescence was expressed as maximum fluorescence minusbaseline fluorescence for each well. The raw data was analyzed againstthe logarithm of test compound concentration by nonlinear regressionwith GraphPad Prism (GraphPad Software, Inc., San Diego, Calif.) usingthe built-in model for sigmoidal dose-response. Antagonist K_(obs)values were determined by Prism using the oxotremorine EC₅₀ value as theK_(D) and the oxotremorine EC₉₀ for the ligand concentration accordingto the Cheng-Prusoff equation (Cheng & Prusoff, 1973).

In this assay, a lower K_(obs) value indicates that the test compoundhas a higher functional activity at the receptor tested.Biphenyl-2-ylcarbamic acid1-[2-(4-{[(R)-2-(3-formylamino-4-hydroxyphenyl)-2-hydroxyethylamino]methyl}-2,5-dimethylphenylcarbamoyl)ethyl]piperidin-4-ylester (compound IIa) was found to have a K_(obs), value of less thanabout 10 nM for blockade of agonist-mediated calcium release in CHOcells stably expressing the hM₁, hM₃ and cM₅ receptors.

Example 15 Whole-cell cAMP Flashplate Assay in HEK-293 and CHO CellLines Heterologously Expressing Human β₁, β₂ or β₃ Adrenergic Receptors

cAMP assays were performed in a radioimmunoassay format using theFlashplate Adenylyl Cyclase Activation Assay System with [¹²⁵I]-cAMP(NEN SMP004, PerkinElmer Life Sciences Inc., Boston, Mass.), accordingto the manufacturers instructions. For the determination of β₁ and β₂receptor agonist potency (EC₅₀), HEK-293 cell lines stably expressingcloned human β₁ and β₂ receptors were grown to near confluency in DMEMsupplemented with 10% FBS and Geneticin (500 μg/mL). For thedetermination β₃ receptor agonist potency (EC₅₀), CHO-K1 cell linestably expressing cloned human or β₃ adrenergic receptors was grown tonear confluency in Hams F-12 media supplemented with 10% FBS andGeneticin (250 μg/mL). Cells were rinsed with PBS and detached in dPBS(Dulbecco's Phosphate Buffered Saline, without CaCl₂ and MgCl₂)containing 2 mM EDTA or Trypsin-EDTA solution (0.05% trypsin/0.53 mMEDTA). After counting cells in Coulter cell counter, cells were pelletedby centrifugation at 1,000 rpm and re-suspended in stimulation buffercontaining IBMX (PerkinElmer Kit) pre-warmed to room temperature to aconcentration of 1.6×10⁶ to 2.8×10⁶ cells/mL. About 40,000 to 80,000cells per well were used in this assay. Test compounds (10 mM in DMSO)were diluted into PBS containing 0.1% BSA in Beckman Biomek-2000 andtested at 11 different concentrations ranging from 100 μM to 1 μM.Reactions were incubated for 10 min at 37° C. and stopped by adding 100μL of cold detection buffer containing [¹²⁵I]-cAMP (NEN SMP004,PerkinElmer Life Sciences, Boston, Mass.). The amount of cAMP produced(pmol/well) was calculated based on the counts observed for the samplesand cAMP standards as described in the manufacturer's user manual. Datawere analyzed by nonlinear regression analysis with the GraphPad PrismSoftware package (GraphPad Software, Inc., San Diego, Calif.) with thesigmoidal equation. The Cheng-Prusoff equation (Cheng Y, and Prusoff WH., Biochemical Pharmacology, 1973, 22, 23, 3099-108) was used tocalculate the EC₅₀ values.

In this assay, a lower EC₅₀ value indicates that the test compound has ahigher functional activity at the receptor tested. Biphenyl-2-ylcarbamicacid1-[2-(4-{[(R)-2-(3-formylamino-4-hydroxyphenyl)-2-hydroxyethylamino]methyl}-2,5-dimethylphenyl-carbamoyl)ethyl]piperidin-4-ylester (compound IIa) was found to have an EC₅₀ value less than about 10nM for the β₂ adrenergic receptor; an EC₅₀ value of about 30 nM for theβ₁ adrenergic receptor; and an EC₅₀ value greater than 700 nM for the 13adrenergic receptor.

Example 16 Whole-Cell cAMP Flashplate Assay With a Lung Epithelial CellLine Endogenously Expressing Human β₂ Adrenergic Receptor

In this assay, the agonist potency and intrinsic activity of a testcompound were determined using a cell line expressing endogenous levelsof the β₂ adrenergic receptor. Cells from a human lung epithelial cellline (BEAS-2B) (ATCC CRL-9609, American Type Culture Collection,Manassas, Va.) (January B, et al., British Journal of Pharmacology,1998, 123, 4, 701-11) were grown to 75-90% confluency in complete,serum-free medium (LHC-9 medium containing epinephrine and retinoicacid, Biosource International, Camarillo, Calif.). The day before theassay, the medium was switched to LHC-8 (no epinephrine or retinoicacid, Biosource International, Camarillo, Calif.). cAMP assays wereperformed in a radioimmunoassay format using the Flashplate AdenylylCyclase Activation Assay System with [¹²⁵I]-cAMP (NEN SMP004,PerkinElmer Life Sciences Inc., Boston, Mass.), according to themanufacturers instructions.

On the day of the assay, cells were rinsed with PBS, lifted by scrapingwith 5 mM EDTA in PBS, and counted. Cells were pelleted bycentrifugation at 1,000 rpm and re-suspended in stimulation bufferpre-warmed to 37° C. at a final concentration of 600,000 cells/mL. Cellswere used at a final concentration of 100,000 to 120,000 cells/well inthis assay. Test compounds were serially diluted into assay buffer (75mM Tris/HCl pH 7.4 at 25° C., 12.5 mM MgCl₂, 1 mM EDTA, 0.2% BSA) inBeckman Biomek-2000. Test compounds were tested in the assay at 11different concentrations, ranging from 10 μM to 10 pM. Reactions wereincubated for 10 min at 37° C. and stopped by addition of 100 μL ofice-cold detection buffer. Plates were sealed, incubated over night at4° C. and counted the next morning in a Topcount scintillation counter(Packard BioScience Co., Meriden, Conn.). The amount of cAMP producedper mL of reaction was calculated based on the counts observed forsamples and cAMP standards, as described in the manufacturer's usermanual. Data were analyzed by nonlinear regression analysis with theGraphPad Prism Software package (GraphPad Software, Inc., San Diego,Calif.) using the 4-parameter model for sigmoidal dose-response.

In this assay, a lower EC₅₀ value indicates that the test compound has ahigher functional activity at the receptor tested. Biphenyl-2-ylcarbamicacid1-[2-(4-{[(R)-2-(3-formylamino-4-hydroxyphenyl)-2-hydroxyethylamino]methyl}-2,5-dimethylphenyl-carbamoyl)ethyl]piperidin-4-ylester (compound IIa) was found to have an EC₅₀ value of less than 10 nMwith intrinsic activity value of greater than 0.3 compared with a fullβ₂ agonist isoproterenol (1.0).

Example 17 Einthoven Assay for Determining Bronchoprotective Efficacyand Duration

In this assay, the bronchoprotective efficacy and duration of testcompounds were determined using guinea pigs. This assay was derived fromthe procedures described in Einthoven (1892) Pfugers Arch. 51: 367-445;and Mohammed et al. (2000) Pulm Pharmacol Ther. 13(6):287-92. In thisassay, changes in ventilation pressure serve as a surrogate measure ofairway resistance. Following pre-treatment with a test compound,muscarinic antagonist potency was determined using bronchoconstrictordose-response curves to intravenous methacholine in the presence ofpropranolol. Similarly, β₂ agonist bronchoprotective potency wasdetermined using histamine. Combined bronchoprotective potency wasdetermined using methacholine in the absence of propranolol.

The assay was conducted using male Duncan-Hartley guinea pigs (Harlan,Indianapolis, Ind.), weighing between 250 and 400 g. A test compound orvehicle (i.e., sterile water) was dosed by inhalation (1H) over a 10minute time period in a whole body exposure dosing chamber (R+S Molds,San Carlos, Calif.) using 5 mL of dosing solution. Animals were exposedto an aerosol generated from an LC Star Nebulizer Set (Model 22F51, PARIRespiratory Equipment, Inc. Midlothian, Va.) driven by Bioblend (amixture of 5% CO₂; 21% O₂; and 74% N₂) at a pressure of 22 psi.Pulmonary function was evaluated at various time points after inhalationdosing.

Seventy-five minutes prior to the start of the assay, the guinea pigswere anesthetized with an intramuscular (IM) injection of a mixture ofketamine (43.7 mg/kg/xylazine (3.5 mg/kg)/acepromazine (1.05 mg/kg). Asupplemental dose of this mixture (50% of initial dose) was administeredas needed. The jugular vein and carotid artery were isolated andcannulated with saline-filled polyethylene catheters (micro-renathaneand PE-50, respectively, Beckton Dickinson, Sparks, Md.). The carotidartery was connected to a pressure transducer to allow the measurementof blood pressure and the jugular vein cannula was used for IV injectionof either methacholine or histamine. The trachea was then dissected freeand cannulated with a 14G needle (#NE-014, Small Parts, Miami Lakes,Fla.). Once the cannulations were complete, the guinea pigs wereventilated using a respirator (Model 683, Harvard Apparatus, Inc., MA)set at a stroke volume of 1 mL/100 g body weight but not exceeding 2.5mL volume, and at a rate of 100 strokes per minute. Ventilation pressure(VP) was measured in the tracheal cannula using a Biopac transducerconnected to a Biopac (TSD 137C) pre-amplifier. Body temperature wasmaintained at 37° C. using a heating pad. Prior to initiating datacollection, pentobarbital (25 mg/kg) was administered intraperitoneally(IP) to suppress spontaneous breathing and obtain a stable baseline. Thechanges in VP were recorded on a Biopac Windows data collectioninterface. Baseline values were collected for at least 5 minutes, afterwhich time guinea pigs were challenged IV non-cumulatively with 2-foldincremental doses of the bronchoconstrictor (methacholine or histamine).When methacholine was used as the bronchoconstrictor agent, animals werepre-treated with propranolol (5 mg/kg, IV) to isolate the antimuscariniceffects of the test compound. The propranolol was administered 30minutes prior to construction of the dose-response curve to methacholineor histamine. Changes in VP were recorded using the Acknowledge DataCollection Software (Santa Barbara, Calif.). After the completion ofstudy, the animals were euthanized.

Change in VP was measured in cm of water. Change in VP (cm H₂O)=peakpressure (after bronchoconstrictor challenge)−peak baseline pressure.The dose-response curve to methacholine or histamine was fitted to afour parameter logistic equation using GraphPad Prism, version 3.00 forWindows (GraphPad Software, San Diego, Calif.). The following equationwas used:

Y=Min+(Max−Min)/(1+10^(((log ID50−X)*Hillslope)))

where X is the logarithm of dose, Y is the response. Y starts at Min andapproaches asymptotically to Max with a sigmoidal shape.

The percent inhibition of the bronchoconstrictor response to asubmaximal dose of methacholine or histamine was calculated at each doseof the test compound using the following equation: % Inhibition ofresponse=100−((peak pressure (after bronchoconstrictor challenge,treated)−peak baseline pressure (treated)*100%/(peak pressure (afterbronchoconstrictor challenge, water)−peak baseline pressure(water)×100). Inhibition curves were fitted using the four parameterlogistic equation from GraphPad software. ID₅₀ (dose required to produce50% inhibition of the bronchoconstrictor response) and Emax (maximalinhibition) were also estimated wherever appropriate.

The magnitude of bronchoprotection at different time-points afterinhalation of the test compound was used to estimate the pharmacodynamichalf-life (PD T_(1/2)). PD T_(1/2) was determined using a non-linearregression fit using a one-phase exponential decay equation (GraphPadPrism, Version 4.00): Y=Span*exp(−K*X)+Plateau; Starts at Span+Plateauand decays to Plateau with a rate constant K. The PD T_(1/2)=0.69/K.Plateau was constrained to 0.

At 1.5 hours post-dose, biphenyl-2-ylcarbamic acid1-[2-(4-{[(R)-2-(3-formylamino-4-hydroxyphenyl)-2-hydroxyethylamino]methyl}-2,5-dimethylphenylcarbamoyl)ethyl]piperidin-4-ylester (IIa) was found to have an ID50 of less than about 50 μg/mL forboth methacholine-induced bronchoconstriction and histamine-inducedbronchoconstriction.

Additionally, this compound produced significant bronchoprotection forup to about 72 hours when administered as a single sub-maximal dose (100μg/mL). In this assay, salmeterol (3 μg/mL) (a β₂ adrenergic receptoragonist) exhibited significant bronchoprotection for 6 to 14 hours; andtiotropium (10 μg/mL) (a muscarinic receptor antagonist) exhibitedsignificant bronchoprotection for greater than 72 hours.

Example 18 Plethysmograph Guinea Pig Assay for DeterminingBronchoprotective Efficacy and Duration

In this assay, the bronchoprotective efficacy and duration of testcompounds was determined using a guinea pig assay.

Groups of 6 male guinea pigs (Duncan-Hartley (HsdPoc:DH) Harlan,Madison, Wis.) weighing between 250 and 350 g were individuallyidentified by cage cards. Throughout the study, animals were allowedaccess to food and water ad libitum. Test compounds were administeredvia inhalation over 10 minutes in a whole-body exposure dosing chamber(R&S Molds, San Carlos, Calif.). The dosing chambers were arranged sothat an aerosol was simultaneously delivered to 6 individual chambersfrom a central manifold. Guinea pigs were exposed to an aerosol of atest compound or vehicle (WFI). The aerosols were generated from aqueoussolutions using an LC Star Nebulizer Set (Model 22F51, PARI RespiratoryEquipment, Inc. Midlothian, Va.) driven by a mixture of gases (CO₂=5%,O₂=21% and N₂=74%) at a pressure of 22 psi. The gas flow through thenebulizer at this operating pressure was approximately 3 L per minute.The generated aerosols were driven into the chambers by positivepressure. No dilution air was used during the delivery of aerosolizedsolutions. During the 10 minute nebulization, approximately 1.8 mL ofsolution was nebulized. This value was measured gravimetrically bycomparing pre- and post-nebulization weights of the filled nebulizer.

The bronchoprotective effects of test compounds administered viainhalation were evaluated using whole body plethysmography at 1.5, 24,48 and 72 hours post-dose. Forty-five minutes prior to the start of thepulmonary evaluation, each guinea pig was anesthetized with anintramuscular injection of ketamine (43.75 mg/kg), xylazine (3.50 mg/kg)and acepromazine (1.05 mg/kg). The surgical site was shaved and cleanedwith 70% alcohol and a 2 to 3 cm midline incision of the ventral aspectof the neck was made. The jugular vein was isolated and cannulated witha saline-filled polyethylene catheter (PE-50, Becton Dickinson, Sparks,Md.) to allow for intravenous infusions of acetylcholine or histamine insaline. The trachea was then dissected free and cannulated with a 14Gteflon tube (#NE-014, Small Parts, Miami Lakes, Fla.). If required,anesthesia was maintained by additional intramuscular injections of theanesthetic mixture. The depth of anesthesia was monitored and adjustedif the animal responded to pinching of its paw or if the respirationrate was greater than 100 breaths per minute.

Once the cannulations were completed, the animal was placed into aplethysmograph (#PLY3114, Buxco Electronics, Inc., Sharon, Conn.) and anesophageal pressure cannula (PE-160, Becton Dickinson, Sparks, Md.) wasinserted to measure pulmonary driving pressure. The teflon tracheal tubewas attached to the opening of the plethysmograph to allow the guineapig to breathe room air from outside the chamber. The chamber was thensealed. A heating lamp was used to maintain body temperature and theguinea pig's lungs were inflated three times with 4 mL of air using a 10mL calibration syringe (#5520 Series, Hans Rudolph, Kansas City, Mo.) toensure that the lower airways had not collapsed and that the animal didnot suffer from hyperventilation.

The pulmonary evaluation was initiated after determining that baselinevalues were within the range of 0.3 to 0.9 mL per cm H₂O for complianceand within the range of 0.1 to 0.199 cm H₂O per mL per second forresistance. A Buxco pulmonary measurement computer program was used forthe collection and derivation of pulmonary values. Starting the programinitiated the experimental protocol and data collection. The changes involume over time that occurred within the plethysmograph with eachbreath were measured via a Buxco pressure transducer. By integratingthis signal over time, a measurement of flow was calculated for eachbreth. This signal and the pulmonary driving pressure changes, collectedusing a Sensym pressure transducer (TRD4100), were connected via a Buxco(MAX 2270) preamplifier to a data collection interface (SFT3400 andSFT3813). All other pulmonary parameters are derived from these twoinputs.

Baseline values were collected for 5 minutes, after which time theguinea pigs were challenged with either acetylcholine or histamine. Whenevaluating the muscarinic antagonist effects of a test compound,propanolol (5 mg/Kg, iv) (Sigma-Aldrich, St. Louis, Mo.) wasadministered 15 minutes prior to challenge with acetylcholine.Acetylcholine (Sigma-Aldrich, St. Louis, Mo.) (0.1 mg/mL) was infusedintravenously for 1 minute from a syringe pump (sp210iw, World PrecisionInstruments, Inc., Sarasota, Fla.) at the following doses and prescribedtimes from the start of the experiment: 1.9 μg/minute at 5 minutes, 3.8μg/minute at 10 minutes, 7.5 μg/minute at 15 minutes, 15.0 μg/minute at20 minutes, 30 μg/minute at 25 minutes and 60 μg/minute at 30 minutes.Alternatively, bronchoprotective effects of the test compound wasassessed in the acetylcholine challenge model without pretreatment withpropanolol.

When evaluating the O₂ adrenergic receptor agonist effects of the testcompound, histamine (25 μg/mL) (Sigma-Aldrich, St. Louis, Mo.) wasinfused intravenously for 1 minute from a syringe pump at the followingdoses and prescribed times from the start of the experiment: 0.5μg/minute at 5 minutes, 0.9 μg/minute at 10 minutes, 1.9 μg/minute at 15minutes, 3.8 μg/minute at 20 minutes, 7.5 μg/minute at 25 minutes and 15μg/minute at 30 minutes. If resistance or compliance did not returned tobaseline values at 3 minutes following each acetylcholine or histaminedose, the guinea pig's lungs were inflated 3 times with 4 mL of air froma 10 mL calibration syringe. The pulmonary parameters recorded includedrespiration frequency (breaths per minute), compliance (mL per cm H₂O)and pulmonary resistance (cm H₂O per mL per second). Once the pulmonaryfunction measurements were completed at minute 35 of this protocol, theguinea pig was removed from the plethysmograph and euthanized by carbondioxide asphyxiation.

The data were evaluated in one of two ways:

(a) Pulmonary resistance (R_(L)) (cm H₂O per mL per second) wascalculated from the ratio of change in pressure to the change in flow.The R_(L) response to acetylcholine (60 μg/min, 1H) was computed for thevehicle and the test compound. The mean acetylcholine response invehicle-treated animals, at each pre-treatment time, was calculated andused to compute percent inhibition of acetylcholine response, at thecorresponding pre-treatment time, at each test compound dose. Inhibitiondose-response curves for ‘R_(L)’ were fitted with a four parameterlogistic equation using GraphPad Prism, version 3.00 for Windows(GraphPad Software, San Diego, Calif.) to estimate bronchoprotectiveID50 (dose required to inhibit the acetylcholine (60 μg/min)bronchoconstriction response by 50%). The following equation was used:

Y=Min+(Max−Min)/(1+10^(((log ID50−X)*Hillslope)))

where X is the logarithm of dose, Y is the response (percent inhibitionof acetylcholine-induced increase in R_(L)). Y starts at Min andapproaches asymptotically to Max with a sigmoidal shape.

(b) The quantity PD₂, which is defined as the amount of acetylcholine orhistamine needed to cause a doubling of the baseline pulmonaryresistance, was calculated using the pulmonary resistance values derivedfrom the flow and the pressure over a range of acetylcholine orhistamine challenges using the following equation (which was derivedfrom an equation used to calculate PC₂₀ values described in AmericanThoracic Society. Guidelines for methacholine and exercise challengetesting—1999. Am J Respir Crit. Care Med. 2000; 161: 309-329):

${PD}_{2} = {{antilog}\left\lbrack {{\log \; C_{1}} + \frac{\left( {{\log \; C_{2}} - {\log \; C_{1}}} \right)\left( {{2\; R_{0}} - R_{1}} \right)}{R_{2} - R_{1}}} \right\rbrack}$

-   -   where:    -   C₁=concentration of acetylcholine or histamine preceding C₂    -   C₂=concentration of acetylcholine or histamine resulting in at        least a 2-fold increase in pulmonary resistance (R_(L))    -   R₀=Baseline R_(L) value    -   R₁═R_(L) value after C₁    -   R₂═R_(L) value after C₂

Statistical analysis of the data was performed using a two tailedStudents t-test. A P-value <0.05 was considered significant.

Compound 50 described in U.S. Patent Publication No. US 2004/0167167A1,published Aug. 24, 2004, was tested in this assay. The chemicalstructure of compound 50 is as follows:

This compound lacks the alkyl groups present on phenyl ring of thecompounds of the present invention. In this assay, compound 50 showed nosignificant bronchoprotection at 24 hours post-dose for doses rangingfrom 3 μg/mL to 300 μg/mL. The PD2x valves for compound 50 at 24 hourswere similar to the vehicle (water) group.

In this assay, salmeterol (100 μg/mL) (a β₂ adrenergic receptor agonist)exhibited significant bronchoprotection for at least 24 hours; andtiotropium (10 μg/mL) (a muscarinic receptor antagonist) exhibitedsignificant bronchoprotection for at least 24 hours.

While the present invention has been described with reference tospecific aspects or embodiments thereof, it will be understood by thoseof ordinary skilled in the art that various changes can be made orequivalents can be substituted without departing from the true spiritand scope of the invention. Additionally, to the extent permitted byapplicable patent statutes and regulations, all publications, patentsand patent applications cited herein are hereby incorporated byreference in their entirety to the same extent as if each document hadbeen individually incorporated by reference herein.

1-25. (canceled)
 26. A method of producing bronchodilation in a mammal,the method comprising administering to the mammal abronchodilation-producing amount of a compound of formula I:

wherein R¹ is methyl or ethyl; R² is methyl or ethyl; or apharmaceutically acceptable salt or stereoisomer thereof.
 27. The methodof claim 26, wherein the compound has formula II:

wherein R¹ is methyl or ethyl; R² is methyl or ethyl; or apharmaceutically acceptable salt thereof.
 28. The method of claim 26,wherein the compound has formula IIa:

or a pharmaceutically acceptable salt thereof.
 29. The method of claim28, wherein the compound or the salt thereof is administered byinhalation.
 30. The method of claim 28, wherein the compound or the saltthereof is in micronized form.
 31. A method of treating chronicobstructive pulmonary disease in a patient, the method comprisingadministering to the patient a compound of formula I:

wherein R¹ is methyl or ethyl; R² is methyl or ethyl; or apharmaceutically acceptable salt or stereoisomer thereof.
 32. The methodof claim 31, wherein the compound has formula II:

wherein R¹ is methyl or ethyl; R² is methyl or ethyl; or apharmaceutically acceptable salt thereof.
 33. The method of claim 31,wherein the compound has formula IIa:

or a pharmaceutically acceptable salt thereof.
 34. A method of treatingasthma in a patient, the method comprising administering to the patienta compound of formula I:

wherein R¹ is methyl or ethyl; R² is methyl or ethyl; or apharmaceutically acceptable salt or stereoisomer thereof.
 35. The methodof claim 34, wherein the compound has formula II:

wherein R¹ is methyl or ethyl; R² is methyl or ethyl; or apharmaceutically acceptable salt thereof.
 36. The method of claim 34,wherein the compound has formula IIa:

or a pharmaceutically acceptable salt thereof.